Abstract:

The present invention concerns methods and compositions related to type 3
phosphodiesterases (PDE3). Certain embodiments concern isolated peptides
corresponding to various PDE3A isoforms and/or site-specific mutants of
PDE3A isoforms, along with expression vectors encoding such isoforms or
mutants. In specific embodiments, methods for identifying
isoform-selective inhibitors or activators of PDE3 are provided, along
with methods of use of such inhibitors or activators in the treatment of
dilated cardiomyopathy, pulmonary hypertension and/or other medical
conditions related to PDE3 effects on cAMP levels in different
intracellular compartments.

Claims:

1. (canceled)

2. The method according to claim 21, wherein the isolated polypeptide is
identical in sequence to SEQ ID NO:1.

3. The method according to claim 21, wherein the isolated polypeptide is
identical in sequence to SEQ ID NO:2.

4. The method according to claim 21, wherein the isolated polypeptide is
identical in sequence to SEQ ID NO:3.

5. The method according to claim 21, wherein the isolated polypeptide has
the sequence of SEQ ID NO:1, with at least one substitution mutation at
serine residues 292, 293, 312 or 438.

6. The method according to claim 5, wherein the substitution mutation
substitutes an alanine or an aspartate residue for the serine residue.

7. The method according to claim 21, wherein the isolated polypeptide has
the sequence of SEQ ID NO:2, with at least one substitution mutation at
serine residues 312 or 438.

8. The method according to claim 7, wherein the substitution mutation
substitutes an alanine or an aspartate residue for the serine residue.

9-20. (canceled)

21. A method of identifying an isoform-selective regulator of PDE3, said
method comprising:(a) obtaining an isolated polypeptide, wherein the
polypeptide has an amino acid sequence that is at least 95% homologous to
the amino acid sequence of SEQ ID NO:1;(b) identifying at least one test
compound that binds to the isolated a first polypeptide;(c) assaying the
at least one test compound for its ability to interfere with binding of
the first polypeptide to PDE3 cAMP or cGMP;(d) assaying the at least one
test compound for its ability to interfere with binding of a second
polypeptide to cAMP or cGMP; and(e) wherein a test compound with the
binding interference of the first polypeptide is greater than the binding
interference to the second polypeptide is an isoform-selective regulator
of PDE3.

22. The method of claim 21, wherein the second polypeptide is a protein
kinase, a phosphatase or a phosphorylase.

23-26. (canceled)

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a divisional of U.S. patent application Ser. No.
10/175,161, filed Jun. 19, 2002, pending, which application claims the
benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent
Application Ser. No. 60/309,271, filed Aug. 1, 2001, the disclosure of
each of which is hereby incorporated herein by this reference in its
entirety.

BACKGROUND OF THE INVENTION

[0003]1. Field of the Invention

[0004]The present invention relates to the field of cardiovascular and
other diseases. More particularly, the present invention concerns
compositions and methods of identification and use of isoform-selective
activators or inhibitors of type 3 phosphodiesterase (PDE3). Other
embodiments of the invention concern high-throughput screening for novel
pharmaceuticals directed against PDE3 isoforms. In certain embodiments,
the compositions and methods disclosed herein are of use for treatment of
cardiomyopathy, pulmonary hypertension and related conditions.

[0005]2. Description of Related Art

[0006]PDE3 cyclic nucleotide phosphodiesterases hydrolyze cAMP and cGMP
and thereby modulate cAMP- and cGMP-mediated signal transduction (Shakur
et al., 2000a). These enzymes have a major role in the regulation of
contraction and relaxation in cardiac and vascular myocytes. PDE3
inhibitors, which raise intracellular cAMP and cGMP content, have
inotropic effects attributable to the activation of cAMP-dependent
protein kinase (PK-A) in cardiac myocytes and vasodilatory effects
attributable to the activation of cGMP-dependent protein kinase (PK-G) in
vascular myocytes (Shakur et al., 2000a). When used in the treatment of
dilated cardiomyopathy, PDE3 inhibitors such as milrinone, enoximone and
amrinone initially elicit favorable hemodynamic responses, but long-term
administration increases mortality by up to 40% (Nony et al., 1994). This
linkage of short-term benefits of PDE3 inhibition to deleterious effects
on long-term survival in dilated cardiomyopathy is one of the most
perplexing problems in cardiovascular therapeutics. However, it is
thought that these biphasic effects reflect the compartmentally
nonselective increases in intracellular cAMP content in cardiac myocytes
current inhibitors display.

[0007]Clinical trials of the use of β-adrenergic receptor agonists,
which, like PDE3 inhibitors, increase intracellular cAMP content in
cardiac myocytes, were terminated prior to completion because of
increased mortality in treated patients, while β-adrenergic receptor
antagonists, which reduce intracellular cAMP content, have been shown to
improve long-term survival despite initially adverse hemodynamic effects.
These findings suggest that both the short-term benefits and long-term
adverse effects of PDE3 inhibition are attributable to increases in
intracellular cAMP content in cardiac myocytes (Movsesian, 1999).

[0009]Another example of cAMP effects is the phosphorylation of
phospholamban, which relieves its inhibition of SERCA2, the
Ca2+-transporting ATPase of the sarcoplasmic reticulum (Simmerman
and Jones, 1998). Ablation of phospholamban in muscle LIM protein
(MLP).sup.-/- mice with dilated cardiomyopathy results in the restoration
of normal chamber size and contractility (Minamisawa et al., 1999),
suggesting that phospholamban phosphorylation may also be beneficial in
cardiomyopathy.

[0010]Other substrates phosphorylated by PK-A may contribute to adverse
effects on long-term survival. Phosphorylation of L-type Ca2+
channels increases their open probability and may be arrhythmogenic
(Fischmeister and Hartzell, 1990), while phosphorylation of proteins in
the mitogen-activated protein kinase (MAP kinase) cascade may alter
myocardial gene transcription so as to speed the progression of the
disease (Cook and McCormick, 1993; Lazou et al., 1994).

[0012]The phosphorylation of individual substrates of PK-A may be
differentially regulated in response to extracellular signals. Evidence
for differential regulation comes from experiments examining the effects
of stimulating adenylate cyclase activity and cAMP formation via
β1-adrenergic, β2-adrenergic or PGE1 receptors.
Activation of β-adrenergic receptors increases cAMP content in both
cytosolic and microsomal fractions of cardiac myocytes and elicits
contractile responses, while activation of PGE1 receptors increases
cytosolic but not microsomal cAMP content and evokes no contractile
response (Hayes et al., 1980; Buxton and Brunton, 1983). Increases in the
amplitude of intracellular Ca2+ transients in response to
β1-adrenergic receptor activation correlate with changes in
microsomal cAMP content and are accompanied by increases in phospholamban
phosphorylation. Conversely, activation of β2-adrenergic
receptors results in an increase in the amplitude of intracellular
Ca2+ transients that does not correlate with changes in microsomal
cAMP content and occurs without increases in phospholamban
phosphorylation (Hohl and Li, 1991; Xiao et al., 1993, 1994). Thus,
activation of different receptors linked to cAMP metabolism can elicit
different responses in cardiac tissues.

[0013]β-adrenergic receptor stimulation and nonselective
phosphodiesterase inhibition have different effects on cAMP-activated
protein phosphorylation in cardiac myocytes (Rapundalo et al., 1989;
Jurvicius and Fischmeister, 1996) that are relevant to the
pathophysiology of dilated cardiomyopathy. In that condition, a
down-regulation of β1-adrenergic receptors and an uncoupling of
β-adrenergic receptor occupancy and adenylate cyclase stimulation
(attributable to increases in β-adrenergic receptor kinase, Gai and
nucleoside diphosphate kinase) contribute to an impairment in cAMP
generation (Movsesian, 1999; Lutz et al., 2001). Studies of cAMP content
in cytosolic and microsomal fractions of failing and non-failing hearts
demonstrate a far greater reduction in cAMP content in microsomal
fractions than in cytosolic fractions of failing myocardium (Bohm et al.,
1994). Taken together, these results indicate that cAMP content in
different intracellular compartments can be selectively regulated to
invoke different responses reflecting the phosphorylation of different
substrates of PK-A. Further, this regulation is altered in dilated
cardiomyopathy.

[0014]Different isoforms of PDE3 are expressed in cardiac and vascular
myocytes and are localized to different intracellular compartments. The
different PDE3 isoforms may differ in their regulation by PK-A and PK-B
(protein kinase B, also known as Akt). PK-B, a downstream effector of
insulin-like growth factors, is an anti-apoptotic mediator in cardiac
myocytes (Fujio et al., 2000; Matsui et al., 1999; Wu et al., 2000). PK-B
may also be involved in proliferative responses in vascular myocytes
(Rocic and Lucchesi, 2001; Duan et al., 2000; Sandirasegarane et al.,
2000). These findings suggest that different PDE3 isoforms may be
involved in cell- and compartment-selective responses to different
signals that have been implicated in the pathophysiology of dilated
cardiomyopathy and/or pulmonary hypertension. Different PDE3 isoforms in
cardiac and vascular myocytes may regulate functionally distinct pools of
cAMP and cGMP involved in the phosphorylation of different substrates of
PK-A and PK-G, and these isoforms may be regulated in response to
different extracellular signals.

[0015]Until the present invention, it was not possible to develop
isoform-selective inhibitors or activators of PDE3 to use in the
treatment of cardiomyopathy and/or pulmonary hypertension.
Isoform-selective PDE3 inhibitors may provide a beneficial effect on
cardiac output without the long-term mortality associated with
non-specific PDE3 inhibitors. Isoform-selective PDE3 activators may have
beneficial anti-apoptotic effects in patients with dilated cardiomyopathy
and/or pulmonary hypertension whose hemodynamic status is not too
compromised to tolerate a reduction in cardiac contractility, without
concomitant arrhythmogenic effects attributable to increases in cytosolic
cAMP content. A paradigm for the latter is the use of β-adrenergic
receptor antagonists in the treatment of dilated cardiomyopathy.

SUMMARY OF THE INVENTION

[0016]Agents capable of selectively activating or inhibiting individual
PDE3 isoforms or of disrupting their intracellular localization may
selectively affect the phosphorylation of smaller subsets of PK-A and
PK-G substrates to therapeutic advantage. Without wishing to be limited
to any one specific embodiment, an agent that selectively inhibits
sarcoplasmic reticulum-associated PDE3A-136 may help to preserve
intracellular Ca2+ cycling and contractility in patients with
dilated cardiomyopathy taking β-adrenergic receptor agonists, which
may reduce arrhythmogenic effects attributable to increases in cytosolic
cAMP content. Alternatively, if the activation of PDE3A-136 by PK-B is
anti-apoptotic in cardiac myocytes, its inhibition may be pro-apoptotic
(possibly explaining the increased long-term mortality seen with PDE3
inhibition in dilated cardiomyopathy), and the selective activation of
this isoform may be desirable. In addition, currently available
competitive PDE3 inhibitors inhibit cAMP activity more potently than they
inhibit cGMP hydrolytic activity, owing to the higher Kms of the
hydrolytic enzymes for cAMP than for cGMP. Agents that inhibit PDE3
activity through other mechanisms, identified by the methods described
herein, may affect hydrolysis of the two substrates differentially,
resulting in different cellular actions of therapeutic benefit.

[0017]As disclosed herein, N-terminal differences exist between the
different isoforms of PDE3. Without wishing to be limited to any one
specific embodiment, these N-terminal differences may offer opportunities
for targeting individual isoforms of PDE3. Differences with respect to
phosphorylation sites that stimulate catalytic activity suggest that
agents that bind to domains containing these sites so as to either block
phosphorylation or mimic its effects may be useful as isoform-selective
PDE3 inhibitors or activators. As an example, an agent that binds to the
P1 phosphorylation site could selectively inhibit or activate PDE3A-136
or PDE3B-137. A similar rationale would apply to agents that bind to
N-terminal protein-interacting domains so as to either block or mimic the
effects of these interactions, with the paradigm of peptides that
modulate cAMP-mediated signaling by blocking PK-A/AKAP interactions
(Rosenmund, et al., 1994). Without wishing to be limited to any one
specific embodiment, the typical accessibility of phosphorylation sites
and protein-interacting domains makes them propitious drug targets.
Differences between PDE3A and PDE3B in the N-terminal regions are
sufficient to permit selective targeting of PDE3A-136 v. PDE3B-137, which
may allow selective modulation of PDE3 activity in cardiac and vascular
myocytes.

[0018]As shown herein, the different isoforms of PDE3 are translated from
different mRNAs. In some cases, these mRNAs are generated from different
genes (PDE3A and PDE3B). In the case of PDE3A, different isoforms are
generated from different mRNAs transcribed from the same gene (e.g.,
PDE3A1 and PDE3A2 mRNAs). The open reading frame (ORF) of PDE3A1 is
indicated in SEQ ID NO:14. The 5' untranslated region (5'-UTR) of PDE3A1,
starting with the first ATG codon, is listed in SEQ ID NO:18. The
approximate ORF of PDE3A2 is indicated in SEQ ID NO:15. A nucleotide
sequence unique to PDE3A1 mRNA has been identified, and cDNA probes have
been designed that react with PDE3A1 mRNA but not PDE3A2 mRNA. Without
wishing to be limited to any one specific embodiment, these differences
make PDE3 mRNAs propititious targets for decreasing the activity of
individual protein isoforms by inhibiting the translation of their mRNAs
via antisense constructs, ribozymes or small interfering RNAs ("siRNAs").

[0019]The present invention fulfills an unresolved need in the art by
identifying differences between PDE3 isoforms that may be used to develop
isoform-selective inhibitors or activators of PDE3 activity. Such
inhibitors or activators are proposed to allow the differential
regulation of cAMP and cGMP levels in different subcellular compartments,
cell types and tissues. In certain embodiments, the present invention
concerns methods for identifying isoform-selective PDE3 inhibitors or
activators. Certain embodiments concern compounds identified by such
methods that are of use for the therapeutic treatment of cardiomyopathy
and/or pulmonary hypertension. In preferred embodiments, such compounds
result in improved cardiac output while exhibiting little or no long-term
toxicity. In other embodiments, the isoform-selective inhibitors or
activators of PDE3 find utility for therapeutic treatment of a number of
disease states related to defects in the regulation of cAMP
concentration, such as diabetes mellitus, peripheral vascular disease and
coronary artery stenosis (especially, but not limited to, stenoses
occurring after coronary angioplasty).

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]The following drawings form part of the present specification and
are included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to one or
more of these drawings in combination with the detailed description of
specific embodiments presented herein.

[0025]FIG. 5A: Western blotting of rtPDE3A1 (containing the full ORF
product of PDE3A1) and microsomal and cytosolic fractions of human
myocardium. A lysate of Sf21 cells expressing a full-length open reading
frame ORF of rePDE3A1 (1.0 μg/lane) and microsomal and cytosolic
fractions of human myocardium (50 and 20 μg/lane, respectively) were
subjected to SDS-PAGE, followed by electrophoretic transfer to
nitrocellulose membranes and Western blotting, using anti-NT, anti-MID
and anti-CT antibodies.

[0026]FIG. 5B: Location of anti-NT, anti-MD and Anti-CT binding sites on
the full-length ORF of PDE3A1.

[0027]FIG. 6: Comparison of molecular weights of [35S]-labeled
rtPDE3A proteins, showing SDS-PAGE autoradiograms, and native cardiac and
aortic isoforms of PDE3A, identified by Western blotting of membranes
prepared from the same gels with antibodies as indicated. The numbers
below the autoradiograms indicate the initial start codon of the
PDE3A-derived construct.

[0028]FIG. 7: Generation of cardiac and aortic isoforms of PDE3A. PDE3A1
and PDE3A2 mRNAs were generated by alternative transcription. PDE3A1 is
expressed only in cardiac myocytes. PDE3A2 is expressed in both cardiac
and aortic myocytes. PDE3A-136 is translated from PDE3A1. PDE3A-118 and
PDE3A-94 are translated from alternative sites in PDE3A2. Numbers in
"mRNA" refer to start codons. P1, P2 and P3 designate phosphorylation
sites.

[0030]FIG. 9: Stimulation of cGMP hydrolytic activity by PK-A.
Detergent-solubilized lysates of Sf21 cells expressing rtPDE3B isoforms
(full-length ORFs, including wild-type, Ala→Ser and
Ser→Asp, mutations) were prepared, and cGMP hydrolytic activity
was determined at 0.03 μM cGMP after incubation in the presence or
absence of PK-A and ATP. Values represent mean±standard deviation
(each pair of values represents data from a single preparation).

[0031]FIG. 10: Co-immunoprecipitation of rtPK-B and rtPDE3B. Amino acid
sequences of rtPDE3B are shown at top. Detergent-solubilized lysates of
Sf9 cells infected with rtPDE3B were mixed with lysates from Sf9 cells
infected with rtPK-B. Proteins were immunoprecipitated with anti-PDE3B
antibodies and subjected to Western blotting with anti-PDE3B and
anti-PK-B antibodies. PK-B co-precipitates with the full-length but not
the truncated rtPDE3B. The identity of the 92 kDa band is unknown.

[0033]FIG. 12: Comparison of apparent molecular weights of [35S]
rtPDE3A proteins and native cardiac and aortic isoforms of PDE3A. rtPDE3A
isoforms were generated by in vitro transcription/translation from
constructs with 5' deletions designed to result in translation from
different in-frame AUG codons in the PDE3A1 ORF.

[0074]As used herein, "a" or "an" may mean one or more than one of an
item.

[0075]This application concerns, at least in part, isolated proteins and
nucleic acids encoded by type 3 phosphodiesterase (PDE3, GenBank
Accession No. NM000921), as well as methods of identification of
isoform-selective inhibitors or activators and methods of therapeutic
treatment of cardiomyopathy and/or pulmonary hypertension directed
towards such proteins. In the present disclosure, reference to "PDE3" or
"type 3 phosphodiesterase," without further qualification or limitation,
means any or all of the isoforms of PDE3, either identified herein or as
discovered or characterized by the methods disclosed herein. Where the
sequences of the disclosed PDE3A isoforms proteins (SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3) differ from the GenBank sequence, the sequences
disclosed herein are believed to be more accurate and are preferred.

[0076]A "PDE3 isoform" is a variant of type 3 phosphodiesterase that
differs in its primary structure (i.e., amino acid sequence) from other
isoforms of PDE3. The term encompasses, but is not limited to, isoforms
that are produced by truncation, amino acid substitution (mutation) or by
alternative mRNA splicing, so long as some difference in amino acid
sequence results. For the purposes of the present invention, other types
of covalent modification would be considered to fall within the scope of
a single isoform. For example, both phosphorylated and unphosphorylated
forms of PDE3A-136 would be considered to represent the same isoform. The
amino acid sequences of the three isoforms of PDE3A are as disclosed in
SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0077]As used herein, an "inhibitor" of PDE3 means any compound or
combination of compounds that acts to decrease the activity of PDE3,
either directly or indirectly, with respect to catalyzing the breakdown
of cAMP and/or cGMP. An inhibitor can be a molecule, an atom, or a
combination of molecules or atoms without limitation. The term
"antagonist" of PDE3 is generally synonymous with an "inhibitor" of PDE3.
Inhibitors may act directly on PDE3 by, for example, binding to and
blocking the catalytic site or some other functional domain of PDE3 that
is required for activity. An inhibitor may also act indirectly, for
example, by blocking the phosphorylation (or its effect on activity) or
facilitating the dephosphorylation of PDE3 or by facilitating or
interfering with the binding of PDE3 to another protein or peptide. The
skilled artisan will realize that inhibitors and/or activators may affect
PDE3 isoform protein activity and/or may affect the transcription,
processing, post-transcriptional modification, stability and/or
translation of one or more mRNA species encoding PDE3 isoform proteins
(see, e.g., GenBank Accession No. NM000921, SEQ ID NO:14, SEQ ID NO:15,
SEQ ID NO:18).

[0078]As used herein, an "activator" of PDE3 means any compound or
combination of compounds that acts to increase the activity of PDE3,
either directly or indirectly, with respect to catalyzing the breakdown
of cAMP and/or cGMP. An activator can be a molecule, an atom, or a
combination of molecules or atoms without limitation. The term "agonist"
of PDE3 is generally synonymous with an "activator" of PDE3. Activators
may act directly on PDE3 by, for example, binding some functional domain
of PDE3 that is required for activity or by altering the secondary,
tertiary or quaternary structure of PDE3 in a way that increases
activity. An activator may also act indirectly, for example, by
facilitating the phosphorylation or mimicking its effect, by blocking the
dephosphorylation of PDE3 or by facilitating or interfering with the
binding of PDE3 to another protein or peptide. As discussed above,
activators may affect PDE3 isoforms at the level of mRNA and/or protein.

[0079]An "isoform-selective" inhibitor or activator of PDE3 is one that
has a greater effect on one isoform of PDE3 than on any other isoform of
PDE3. In preferred embodiments, an "isoform-selective" inhibitor or
activator has at least a two-fold greater, more preferably three-fold
greater, even more preferably four-fold greater, yet more preferably
five-fold, and most preferably ten-fold or more greater effect on one
isoform of PDE3 than on any other isoform of PDE3. For purposes of the
present invention, the precise degree of selectivity of an inhibitor or
activator for one isoform of PDE3 compared to other isoforms is not
significant, so long as a desired therapeutic effect is achieved. For
example, a desired therapeutic effect might be an improvement in cardiac
output, with a decrease in long-term mortality, resulting from
administration of an isoform-selective PDE3 inhibitor or activator
compared with nonspecific PDE3 inhibitors. An "isoform-selective"
inhibitor or activator of PDE3 encompasses, but is not limited to, an
isoform-specific inhibitor or activator of PDE3. An isoform-specific
inhibitor or activator of PDE3 is one that acts almost exclusively upon a
single isoform of PDE3, so that the effect of the inhibitor or activator
on one isoform of PDE3 compared to any other PDE3 isoform is at least an
order of magnitude greater, more preferably two orders of magnitude
greater, and most preferably three orders of magnitude or more greater.

Type 3 Phosphodiesterase

[0080]Cyclic nucleotide phosphodiesterases have a ubiquitous role in
regulating cAMP- and cGMP-mediated intracellular signaling. Eleven
families of these enzymes have been identified. Those in the PDE3 family
are dual-specificity phosphodiesterases that bind both cAMP and cGMP with
high affinity and hydrolyze them in a mutually competitive manner (FIG.
1). PDE3 inhibitors, which raise intracellular cAMP and cGMP content,
have inotropic effects attributable to the activation of cAMP-dependent
protein kinase (PK-A) in cardiac myocytes and vasodilatory effects
attributable to the activation of cGMP-dependent protein kinase (PK-G) in
vascular myocytes.

[0082]Two subfamilies of PDE3, products of genes designated PDE3A and
PDE3B, have been identified. PDE3A is expressed primarily in cardiac and
vascular myocytes and platelets, while PDE3B is expressed primarily in
adipocytes, hepatocytes and pancreatic cells (but also in vascular
myocytes) (Reinhardt et al., 1995). To date, one PDE3B (Taira et al.,
1993) and three PDE3A cDNAs have been cloned. The latter are generated by
transcription from alternative start sites in PDE3A. PDE3A1 (SEQ ID
NO:14, SEQ ID NO:18), which was cloned from human myocardium,
incorporates all sixteen exons of PDE3A (Meacci et al., 1992; Kasuya et
al., 2000). PDE3A2 (SEQ ID NO:15), which was cloned from aortic myocytes,
is transcribed from a start site in exon 1 (Choi et al., 2001). PDE3A3,
cloned from placenta, is transcribed from a start site between exons 3
and 4 (Kasuya et al., 1995). The alternative start sites used for
transcription of the three PDE3A mRNAs are illustrated in FIG. 3. The
encoded amino acid sequences of the PDE3A isoforms are disclosed herein
as SEQ ID NO:1 (PDE3A-136), SEQ ID NO:2 (PDE3A-118) and SEQ ID NO:3
(PDE3A-94). The skilled artisan will realize that the protein isoforms of
PDE3 do not precisely correspond to the mRNA species transcribed from the
PDE3A gene. For example, both PDE3A-118 and PDE3A-94 are translated from
the PDE3A2 mRNA (SEQ ID NO:15).

[0083]The functional topographies of the proteins corresponding to the
longest open reading frames (ORFS) of PDE3A and PDE3B are similar (FIG.
4). The C-terminus includes a sequence of about 280 amino acids,
designated as "CCR" (FIG. 4), which is highly conserved among cyclic
nucleotide phosphodiesterase families and in which catalytic activity
resides. Within CCR lies a 44-amino acid insert, designated "INS," that
is unique to the PDE3 family of cyclic nucleotide phosphodiesterases. The
N-terminus contains two hydrophobic sequences, designated "NHR1" (about
200 amino acids) and "NHR2" (about 50 amino acids). NHR1 and NHR2 appear
to be implicated in intracellular targeting. Between NHR1 and NHR2 are
sites phosphorylated by PK-A and PK-B that, despite their distance from
CCR, modulate catalytic activity. A second PK-A site whose function is
unclear is located between NHR2 and CCR.

[0084]Despite the structural similarities, there are considerable
differences between PDE3A and PDE3B with respect to their amino acid
sequences. PDE3A and PDE3B are 84 to 86% identical within the CCR region,
exclusive of INS. However, INS and the extreme C-terminus are only 35 to
39% identical, and the remaining upstream regions are less than 30%
identical. Thus, while the catalytic sequences of the isoforms are
similar, the regulatory portions of the isoforms appear to be very
different and are likely to be differentially affected by the various
inhibitors and activators of the present invention.

[0085]Structure/Function Relations

[0086]Catalytic activity. The catalytic activity of PDE3 enzymes requires
almost the entire C-terminal sequence downstream of about amino acid 650,
including the CCR domain that is largely conserved among all PDE
families, as well as the INS and the CCR-flanking regions that are unique
to the PDE3 family (FIG. 4). (Cheung et al., 1996; He et al., 1998.) The
recent determination of the crystal structure of the related enzyme
PDE4B2B has led to the identification of its catalytic site (Xu et al.,
2000). The catalytic domain consists of three subdomains comprising 17
α-helices.

[0087]The active site, preserved in all PDE families, is at the junction
of these three subdomains and is formed by the apposition of
discontinuous amino acids. Differences in substrate affinity and
selectivity among isoform families may be influenced in large part by
differences in amino acid sequences that allosterically affect Glu1001 of
PDE3A, which "reads" the 1- and 6-positions of the cyclic nucleotide
purine ring and determines affinity (and, hence, selectivity) for cAMP
and cGMP. Experiments involving PDE3/PDE4 chimeras indicate that the
regions adjacent to this site contain the determinants of sensitivity to
phosphodiesterase inhibitors (Atienza et al., 1999). This model, in which
the active site is formed by discontinuous domains with allosteric
determination of substrate affinity, may explain why so much sequence is
required for catalytic activity. It may also explain why mutations of
some amino acids preferentially affect binding of either cAMP or cGMP,
while others affect the binding of both nucleotides (Zhang and Colman,
2000). While the N-terminus is not required for catalytic activity,
N-terminal deletions increase the ratio of Vmax cGMP/Vmax cAMP,
suggesting that the N-terminal region is involved in regulating catalytic
activity (Tang et al., 1997).

[0088]The structural model described above has important implications
regarding the feasibility of selective PDE3 inhibition or activation. The
sequences of regions required for catalytic activity, INS and the regions
flanking CCR, differ sufficiently between PDE3A and PDE3B to be
reasonable targets for isoform-selective inhibitors or activators. As
described in the Examples below, the development of anti-peptide
antibodies selective for the C-terminus of either PDE3A or PDE3B is
further evidence that selective inhibition or activation may occur. The
existence of allosteric sites that differentially affect cAMP and cGMP
hydrolysis allows for the identification of small molecules that
selectively bind to these sites and affect either cAMP or cGMP
hydrolysis.

[0089]Intracellular localization. Intracellular targeting of PDE3 appears
to be determined principally by the N-terminal domains NHR1 and NHR2.
NHR1 contains six transmembrane helices, the last two of which are
sufficient to localize recombinant proteins containing these domains
exclusively to intracellular membranes (Kenan et al., 2000; Shakur et
al., 2000b). Such recombinants can be solubilized only by a combination
of high salt and detergent, suggesting that they are intrinsic membrane
proteins. Recombinants lacking NHR1 but retaining NHR2 are found in both
microsomal and cytosolic fractions of transfected cells. High salt alone
is sufficient to solubilize these proteins, suggesting that interactions
with other proteins are involved in their intracellular localization.
Recombinants lacking both NHR1 and NHR2 are predominantly cytosolic.

[0090]Regulation by protein phosphorylation. Phosphorylation of PDE3 plays
a major role in the regulation of its function. In adipocytes,
phosphorylation of PDE3 by PK-A and perhaps PI3-K are involved in the
anti-lipolytic response to insulin (Smith et al., 1991). In oocytes,
phosphorylation by PK-B results in the resumption of meiosis (Zhao et
al., 1998). In promyeloid cells, phosphorylation by PK-B regulates cAMP
pools that modulate DNA synthesis (Ahmad et al., 2000). In platelets,
phosphorylation of PDE3A by PK-A and an insulin-activated protein kinase
is associated with inhibition of aggregation (Grant et al., 1988;
Lopez-Aparicio et al., 1993).

[0091]As described in more detail in the Examples below, three
phosphorylation sites have been identified for the PDE3 isoforms (FIG.
4). PDE3B is phosphorylated in vivo by PK-A and possibly by PI3-K at
Ser318 (site P2) (Rahn et al., 1996; Rondinone et al., 2000). The P2 site
is dephosphorylated by a PP2A serine/threonine phosphatase (Resjo et al.,
1999). PDE3B is also phosphorylated in vivo by PK-B at Ser296 (site P1)
(Kitamura et al., 1999). Phosphorylation at either site increases
catalytic activity. The fact that P1 and P2 lie between NHR1 and NHR2
raises the possibility that phosphorylation at these sites also affects
intracellular targeting.

[0092]A third site, Ser421 in PDE3B (site P3), is phosphorylated by PK-A
in vitro (Rascon et al., 1994). In adipocytes, it is unclear whether
PDE3B is phosphorylated at P3 in response to isoproterenol or insulin in
vivo. It is unknown whether this site is phosphorylated in PDE3B in other
cell types and, if so, how phosphorylation at this site affects activity.
It is also unknown whether phosphorylation at any of these sites affects
inhibitor sensitivity, but a relevant paradigm is the reduction in the
sensitivity of another phosphodiesterase, PDE4D3, to the inhibitor
rolipram that results from phosphorylation of PDE4D3 by PK-A (Hoffmann et
al., 1998). Prior to the present invention, the phosphorylation sites on
the PDE3A isoforms were unknown. Numerous consensus phosphorylation sites
are present in the PDE3A amino acid sequence and it was unknown which of
these sites was phosphorylated in vivo.

[0093]The identification of protein kinases that phosphorylate PDE3
isoforms and alter their function may elucidate their role in dilated
cardiomyopathy. Phosphorylation and activation of PDE3 by PK-B, for
example, may be an anti-apoptotic mechanism related to the deleterious
long-term effects of PDE3 inhibition in dilated cardiomyopathy. The
sequences of PDE3A and PDE3B contain multiple consensus sites for CK2,
PK-C and other protein kinases. It may be especially important to
consider cross-regulation by these kinases in the pathophysiology of
cardiomyopathy and/or pulmonary hypertension. By analogy, PDE4D3
phosphorylation by ERK2 profoundly reduces its activity, and this
reduction is reversed by phosphorylation by PK-A (Hoffmann et al. 1999).

[0094]Protein-Protein Interactions

[0095]Interactions with other proteins are involved in the regulation of
activity and intracellular localization of other families of PDE. Binding
of Ca2+/CaM stimulates catalytic activity of PDE1 via multiple
CaM-binding domains (Sonnenburg et al., 1995). The activities of PDE6
αβ and α'α' dimers are inhibited by their
interaction with PDEγ. Phototransduction occurs when this
inhibition is relieved by interaction with the rhodopsin-coupled G
protein transducin (Granovsky et al., 2000). PDE6 dissociates from
intracellular membranes upon binding to PDEδ (Florio et al., 1996).
Interactions with RACK1 and AKAPs are involved in the subcellular
targeting of PDE4 isoforms to multi-enzyme complexes (Yarwood et al.,
1999; Dodge et al., 2001). The interactions of PDE4 with SH3 domains of
SRC family tyrosine kinases affect intracellular localization and
inhibitor sensitivity (McPhee et al., 1999).

[0096]Prior to the present invention, it was unknown whether PDE3 is
catalytically regulated or intracellularly targeted via interactions with
other proteins. PDE3B, insulin receptor, the p85 and p110 subunits of
PI3-K and an unidentified 97-kDa protein are co-immunoprecipitated from
human adipocytes with anti-insulin receptor antibodies (Rondinone et al.,
2000). Preliminary data on the interaction of PDE3B with 14-3-3 proteins
has been reported (Palmer et al., 2000). 14-3-3 proteins bind to
phosphorylated serine residues in consensus motifs and affect
intracellular localization of proteins in diverse ways (Fu et al., 2000).
As discussed in the Examples below, site P1 in PDE3A and PDE3B
approximates this consensus motif, raising the possibility that
phosphorylation affects intracellular localization through interaction
with 14-3-3 proteins. The Examples further show the existence of stable
complexes of PDE3B with PK-B and AKAP220. Taken together, these
observations indicate that interactions of other proteins with the
N-terminus are involved in PDE3 function, and that phosphorylation of
PDE3 may affect these interactions.

Proteins

[0097]In referring to the function of PDE3 or "wild-type" activity, it is
meant that the molecule in question has the ability to catalyze the
breakdown of cAMP and cGMP. Molecules possessing this activity may be
identified using assays familiar to those of skill in the art. For
example, in vitro assay of homogenates containing PDE3 activity, or
variants thereof, will identify those molecules having PDE3 activity by
virtue of their ability to degrade cAMP or cGMP. The skilled artisan will
realize that a variety of phosphodiesterases are endemic to various cell
lines and tissues and will select an appropriate system lacking
endogenous phosphodiesterase to perform such assays.

[0098]The term "PDE3 gene" refers to any DNA sequence that is
substantially identical to a DNA sequence encoding a PDE3 protein as
defined above. Allowing for the degeneracy of the genetic code, sequences
that have at least about 50%, usually at least about 60%, more usually
about 70%, most usually about 80%, preferably at least about 90%, more
preferably at least about 95%, most preferably 98% or more of nucleotides
that are identical to the cDNA sequences of PDE3 are "as set forth in"
those sequences. Sequences that are substantially identical or
"essentially the same" as the cDNA sequences of PDE3 also may be
functionally defined as sequences that are capable of hybridizing to a
nucleic acid segment containing the complement of the cDNA sequences of
PDE3 under conditions of relatively high stringency. Such conditions are
typically relatively low salt and/or high temperature conditions, such as
provided by about 0.02 M to about 0.15 M NaCl at temperatures of about
50° C. to about 70° C. Such selective conditions tolerate
little, if any, mismatch between the complementary strands and the
template or target strand. Any such gene sequences may also comprise
associated control sequences.

[0100]Substantially identical analog proteins will be greater than about
80% identical, more preferably 90% identical, even more preferably 95%
identical, yet more preferably 98% identical, even more preferably 99%
identical, yet even more preferably 99.5% identical, and most preferably
99.9% identical to the corresponding sequence of the native protein.
Sequences having lesser degrees of similarity but comparable biological
activity are considered to be equivalents. In determining nucleic acid
sequences, all subject nucleic acid sequences capable of encoding
substantially similar amino acid sequences are considered to be
substantially similar to a reference nucleic acid sequence, regardless of
differences in codon sequence.

[0101]Protein Purification

[0102]Certain embodiments may involve purification of one or more
individual PDE3 isoforms or variants thereof. Protein purification
techniques are well known to those of skill in the art. These techniques
involve, at one level, the crude fractionation of the cellular milieu to
polypeptide and non-polypeptide fractions. Having separated the
polypeptide from other proteins, the polypeptide of interest may be
further purified using chromatographic and electrophoretic techniques to
achieve partial or complete purification (or purification to
homogeneity). Analytical methods particularly suited to the preparation
of a pure peptide are ion-exchange chromatography, gel exclusion
chromatography, polyacrylamide gel electrophoresis, affinity
chromatography, immunoaffinity chromatography and isoelectric focusing. A
particularly efficient method of purifying peptides is fast protein
liquid chromatography (FPLC) or even HPLC.

[0103]Certain aspects of the present invention concern the purification,
and in particular embodiments, the substantial purification, of an
encoded protein or peptide. The terms "isolated" or "purified" as applied
to a protein or peptide, are intended to refer to a composition,
isolatable from other components, wherein the protein or peptide is
purified to any degree relative to its naturally obtainable state. A
purified protein or peptide, therefore, also refers to a protein or
peptide free from the environment in which it may naturally occur.

[0104]Generally, "purified" will refer to a protein or peptide composition
that has been subjected to fractionation to remove various other
components, and which composition substantially retains its expressed
biological activity. Where the term "substantially purified" is used,
this designation will refer to a composition in which the protein or
peptide forms the major component of the composition, such as
constituting about 50%, about 60%, about 70%, about 80%, about 90%, about
95%, about 98%, about 99% or more of the proteins in the composition.

[0105]Various methods for quantifying the degree of purification of a
protein or peptide will be known to those of skill in the art. These
include, for example, determining the specific activity of an active
fraction or assessing the amount of polypeptides within a fraction by
SDS/PAGE analysis. A preferred method for assessing the purity of a
fraction is to calculate the specific activity of the fraction, to
compare it to the specific activity of the initial extract, and to thus
calculate the degree of purity therein, assessed by a "-fold purification
number." The actual units used to represent the amount of activity will,
of course, be dependent upon the particular assay technique chosen to
follow the purification, and whether or not the protein or peptide
exhibits a detectable activity.

[0106]Various techniques suitable for use in protein purification will be
well known to those of skill in the art. These include, for example,
precipitation with ammonium sulphate, PEG, antibodies and the like, or by
heat denaturation, followed by: centrifugation; chromatography steps such
as ion exchange, gel filtration, reverse phase, hydroxylapatite and
affinity chromatography; isoelectric focusing; gel electrophoresis; and
combinations of these and other techniques. As is generally known in the
art, it is believed that the order of conducting the various purification
steps may be changed, or that certain steps may be omitted, and still
result in a suitable method for the preparation of a substantially
purified protein or peptide.

[0107]There is no general requirement that the proteins or peptides always
be provided in their most purified state. Indeed, it is contemplated that
less substantially purified products will have utility in certain
embodiments. Partial purification may be accomplished by using fewer
purification steps in combination, or by utilizing different forms of the
same general purification scheme. For example, it is appreciated that a
cation-exchange column chromatography performed utilizing an HPLC
apparatus will generally result in a greater "-fold" purification than
the same technique utilizing a low-pressure chromatography system.
Methods exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product or in maintaining the
activity of an expressed protein.

[0108]It is known that the migration of a polypeptide can vary, sometimes
significantly, with different conditions of SDS/PAGE (Capaldi et al.,
1977). It will, therefore, be appreciated that under differing
electrophoresis conditions, the apparent molecular weights of purified or
partially purified expression products may vary.

[0109]High Performance Liquid Chromatography (HPLC) is characterized by a
very rapid separation with high resolution of peaks. Moreover, only a
very small volume of the sample is needed because the particles are so
small and close-packed that the void volume is a very small fraction of
the bed volume. Also, the concentration of the sample need not be very
great because the bands are so narrow that there is very little dilution
of the sample.

[0110]Gel chromatography, or molecular sieve chromatography, is a type of
partition chromatography that is based on molecular size. As long as the
material of which the particles are made does not adsorb the molecules,
the sole factor determining rate of flow is the size of the pores. Hence,
molecules are eluted from the column in decreasing size, so long as the
shape is relatively constant. In gel chromatography, separation is
independent of all other factors such as pH, ionic strength, temperature,
etc.

[0111]Affinity chromatography relies on the specific affinity between a
substance to be isolated and a molecule to which it can specifically
bind. The column material is synthesized by covalently coupling one of
the binding partners, such as an antibody or an antibody-binding protein
to an insoluble matrix. The column material is then able to specifically
adsorb the target substance from the solution. Elution occurs by changing
the conditions to those in which binding will not occur (e.g., altered
pH, ionic strength, temperature, etc.). One of the most common forms of
affinity chromatography is immunoaffinity chromatography. The generation
of antibodies that would be suitable for use in accord with the present
invention is discussed below.

[0112]Synthetic Peptides

[0113]In some embodiments, the present invention concerns smaller peptides
for various uses, such as antibody generation or screening for potential
inhibitors or activators that can bind to various epitopes of PDE3.
Smaller peptides of about 100 amino acids or less can be synthesized in
solution or on a solid support in accordance with conventional
techniques. Various automated peptide synthesizers are commercially
available and can be used in accordance with known protocols. See, for
example, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986);
and Barany and Merrifield (1979), each incorporated herein by reference.
Short peptide sequences, or libraries of overlapping peptides, usually
from about 6 up to about 35 to 50 amino acids, which correspond to
selected regions of the PDE3 protein, can be readily synthesized and then
screened in screening assays designed to identify reactive peptides or
other small molecules. Alternatively, recombinant DNA technology may be
employed wherein a nucleotide sequence which encodes a peptide of the
invention is inserted into an expression vector, transformed or
transfected into an appropriate host cell, and cultivated under
conditions suitable for expression. Expression of cloned PDE3 sequences
is preferred in embodiments where PDE3 peptides of greater than about 50
amino acids in length are desired. The skilled artisan will realize that
it is also possible to synthesize short peptide fragments and covalently
link them together, for example, using carbodiimides as cross-linking
groups. In this manner, a peptide of any desired length can be produced
by synthesizing shorter fragments and joining them in the appropriate
order.

[0114]Two-Dimensional Mapping

[0115]Two-dimensional mapping, also known as proteome analysis, is a
useful tool for characterization of cellular protein expression.
Specifically contemplated are the methods described in Gibson (1974);
Beemon and Hunter (1978); and Luo, et al. (1990), each of which is
incorporated herein by reference in their entirety. Two-dimensional
mapping is based on two-dimensional electrophoretic separation of
proteins in a cellular lysate or homogenate so that each protein can be
identified using specific coordinates in a two-dimensional protein map
from which it can be extracted and further identified (by, e.g., micro
sequencing or mass spectrometry).

[0116]For mapping, the proteins in a cellular homogenate or lysate are
immunoprecipitated, using an antibody or series of antibodies specific
for the proteins of interest, and run on a preparative electrophoretic
protein gel. The proteins from this gel are then transferred to an
immobilizing matrix. Various immobilizing matrices are available and may
be used. Preferred matrices for purposes of the present invention are
nitrocellulose or a nylon matrix such as Immobilon (Millipore, Bedford,
Mass.). The resulting protein-matrix hybrid, called a blot, is then
washed with water in order to remove any non-bound cellular debris from
the initial homogenate or lysate, which may cause interference in
subsequent steps. The blot is then contacted with an antibody, or series
of antibodies, specific to the protein or proteins of interest in the
cellular homogenate or lysate. The skilled artisan will realize that
these antibodies may be monoclonal, polyclonal, or both and use of any
will not substantially change the outcome of this procedure. Once the
protein or proteins of interest from the cellular homogenate or lysate
are identified by the antibodies binding to the proteins and forming an
antibody-protein complex, they are physically excised from the rest of
the blot matrix. One of reasonable skill in the art will recognize that
any common method of antibody detection may be used to identify the
aforementioned antibody-protein complex. These may include, but are not
limited to, ELISA, alkaline-phosphatase-conjugated secondary antibody,
enzyme-conjugated antibodies, radiolabeled antibodies, or any other
common method of detection. For purposes of the present invention,
radiolabeled antibodies are the preferred method of detection.

[0117]The protein or proteins, still in the form of bands from the
immobilizing matrix, are digested by one of several common peptidase
enzymes. These are enzymes that cleave proteins at specific locations
only and include, but are not limited to, trypsin, chymotrypsin, CNBr and
V8. Digestion may be allowed to run to completion, i.e., where every
possible site that the chosen peptidase could recognize in the sample is
cleaved, or it may be a partial digestion, merely run for a shorter
period of time and not to completion. Once the desired level of digestion
is completed, the peptidase chosen is removed from the sample, typically
by centrifugation and transfer of the supernatant to a new container or
vessel.

[0118]These digested samples are then loaded onto a cellulose thin layer
plate for pH-driven electrophoresis, the first "dimension" in the mapping
process. The digested proteins will behave on this thin layer plate much
as they would when subjected to standard SDS-PAGE, except that the
digested protein fragments will separate by charge according to the pH of
the electrophoresis buffer. By way of example only, if the
electrophoresis buffer chosen has a pH ranging from 1.9 to 4.72, then the
majority of the digested peptide fragments in the sample will be
positively charged. The thin layer plate should thus be loaded
appropriately for optimal separation of the digested peptide fragments.
In this example, the plate should be loaded at a distance closer to the
positive electrode and farther from the negative electrode. The skilled
artisan will recognize that the pH used in any individual electrophoresis
should be that which will give an optimal distribution of the peptides.
Preferred pH values include 8.9, more preferably 4.72, even more
preferably 1.9. After electrophoresis is complete, the thin layer plate
is typically dried in an oven. It is thought that this step irreversibly
binds the digested peptide fragments to the cellulose on the thin layer
plate.

[0119]Chromatography, the second "dimension" in the mapping, is next
performed. The thin layer plate is placed in a chamber with a
chromatography liquid, but only one side of the thin layer plate is
immersed in this liquid. The thin layer plate should be placed in the
liquid in such a manner that the liquid used, as it travels up through
the thin layer plate via capillarity, does so at a ninety (90) degree
angle from the direction electrophoresis was performed on the plate. When
chromatography is performed in this way, it will separate the digested
peptide fragments in some manner apart from overall charge. Thus, when
chromatography has completed, the digested peptides will have been
separated first by overall charge, then by a property driven by the
chromatography liquid, hence the "two-dimensional" separation.

[0120]The skilled artisan will recognize that the chromatography buffer
will differ based upon the desired property for separation and will use
that buffer that will give optimal separation of the peptides in
question. By way of example only, chromatography buffers may be selected
that separate according to hydrophobicity, alkalinity, water solubility,
or any other common means of separation apart from overall charge.

[0121]Once chromatography is complete, the thin layer plate is dried and
the digested peptide fragments thus separated are detected using common
means (such as detection of a radioactively labeled antibody).

Protein Chips

[0122]Protein chip technology provides a means of rapidly screening sample
compounds for their ability to hybridize to PDE3 isoform proteins,
peptides or subunits immobilized on a solid substrate. Specifically
contemplated are protein array-based technologies such as those disclosed
by Cheng et al. (U.S. Pat. No. 6,071,394), Zanzucchi et al. (U.S. Pat.
No. 5,858,804) and Lee et al. (U.S. Pat. No. 5,948,627), each of which is
incorporated herein by reference in their entirety. These techniques
involve methods for analyzing large numbers of samples rapidly and
accurately. The technology capitalizes on the binding properties of
proteins or peptides to screen samples.

[0123]A protein chip or array consists of a solid substrate upon which an
array of proteins or peptides have been attached. For screening, the chip
or array is contacted with a sample containing one or more test compounds
that may function as PDE3 inhibitors or activators. The degree of
stringency of binding of test compound to peptides may be manipulated as
desired by varying, for example, salt concentration, temperature, pH and
detergent content of the medium. The chip or array is then scanned to
determine which proteins or peptides have bound to a test compound.

[0124]The structure of a protein chip or array comprises: (1) an
excitation source; (2) an array of probes; (3) a sampling element; (4) a
detector; and (5) a signal amplification/treatment system. A chip may
also include a support for immobilizing the probe.

[0125]In particular embodiments, a protein or peptide may be tagged or
labeled with a substance that emits a detectable signal. The tagged or
labeled species may be fluorescent, phosphorescent, or luminescent, or it
may emit Raman energy or it may absorb energy. When the protein or
peptide binds to a test compound, a signal is generated that is detected
by the chip. The signal may then be processed in several ways, depending
on the nature of the signal. In alternative embodiments, the test
compounds may be labeled.

[0126]The proteins or peptides may be immobilized onto an integrated
microchip that also supports a phototransducer and related detection
circuitry. Alternatively, PDE3 proteins or peptides may be immobilized
onto a membrane or filter that is then attached to the microchip or to
the detector surface itself. The proteins or peptides may be directly or
indirectly immobilized onto a transducer detection surface to ensure
optimal contact and maximum detection. A variety of methods have been
utilized to either permanently or removably attach proteins to a
substrate. When immobilized onto a substrate, the proteins are stabilized
and may be used repeatedly.

[0128]Binding of proteins or peptides to a selected support may be
accomplished by any of several means. For example, proteins may be bound
to glass by first silanizing the glass surface, then activating with
carbodiimide or glutaraldehyde. Alternative procedures may use reagents
such as 3-glycidoxypropyltrimethoxysilane (GOP) or
aminopropyltrimethoxysilane (APTS) linked via amino groups. With
nitrocellulose membranes, the protein probes may be spotted onto the
membranes.

[0129]Specific proteins or peptides may first be immobilized onto a
membrane and then attached to a membrane in contact with a transducer
detection surface. This method avoids binding the protein onto the
transducer and may be desirable for large-scale production. Membranes
particularly suitable for this application include nitrocellulose
membrane (e.g., from BioRad, Hercules, Calif.) or polyvinylidene
difluoride (PVDF) (BioRad, Hercules, Calif.) or nylon membrane
(Zeta-Probe, BioRad) or polystyrene base substrates (DNA.BIND® Costar,
Cambridge, Mass.).

Antibodies

[0130]Antibody Production

[0131]Certain embodiments of the present invention involve antibody
production against one or more PDE3 isoforms. Means for preparing and
characterizing antibodies are well known in the art (see, e.g, Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988, incorporated herein by reference).

[0132]Methods for generating polyclonal antibodies are well known in the
art. Briefly, a polyclonal antibody is prepared by immunizing an animal
with an immunogenic composition and collecting antisera from that
immunized animal. A wide range of animal species may be used for the
production of antisera. Typically, the animal used for production of
anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a
goat. Because of the relatively large blood volume of rabbits, a rabbit
is a preferred choice for production of polyclonal antibodies.

[0133]As is well known in the art, a given composition may vary in its
immunogenicity. It is often necessary, therefore, to boost the host
immune system, as may be achieved by coupling a peptide or polypeptide
immunogen to a carrier. Exemplary and preferred carriers are keyhole
limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins
such as ovalbumin, mouse serum albumin or rabbit serum albumin may also
be used as carriers. Means for conjugating a polypeptide to a carrier
protein are well known in the art and include glutaraldehyde,
m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and
bis-biazotized benzidine.

[0134]As is also well known in the art, the immunogenicity of a particular
immunogen composition may be enhanced by the use of non-specific
stimulators of the immune response, known as adjuvants. Exemplary and
preferred adjuvants include complete Freund's adjuvant (a non-specific
stimulator of the immune response containing killed Mycobacterium
tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide
adjuvant.

[0135]The amount of immunogen composition used in the production of
polyclonal antibodies varies upon the nature of the immunogen as well as
the animal used for immunization. A variety of routes may be used to
administer the immunogen (subcutaneous, intramuscular, intradermal,
intravenous and intraperitoneal). The production of polyclonal antibodies
may be monitored by sampling blood of the immunized animal at various
points following immunization. Later, booster injections may also be
given. The process of boosting and titering is repeated until a suitable
titer is achieved. When a desired level of immunogenicity is obtained,
the immunized animal may be bled and the serum isolated and stored,
and/or the animal may be used to generate MAbs. For production of rabbit
polyclonal antibodies, the animal may be bled through an ear vein or
alternatively by cardiac puncture. The removed blood is allowed to
coagulate and then centrifuged to separate serum components from whole
cells and blood clots. The serum may be used as is for various
applications or else the desired antibody fraction may be purified by
well-known methods, such as affinity chromatography using another
antibody or a peptide bound to a solid matrix.

[0136]Monoclonal antibodies (MAbs) may be readily prepared through use of
well-known techniques, such as those exemplified in U.S. Pat. No.
4,196,265, incorporated herein by reference. Typically, this technique
involves immunizing a suitable animal with a selected immunogen
composition, e.g., a purified or partially purified expressed protein,
polypeptide or peptide. The immunizing composition is administered in a
manner effective to stimulate antibody producing cells.

[0137]The methods for generating monoclonal antibodies (MAbs) generally
begin along the same lines as those for preparing polyclonal antibodies.
Rodents such as mice and rats are preferred animals, however, the use of
rabbit, sheep or frog cells is also possible. Following immunization,
somatic cells with the potential for producing antibodies, specifically B
lymphocytes (B cells), are selected for use in the MAb-generating
protocol. These cells may be obtained from biopsied spleens, tonsils or
lymph nodes, or from a peripheral blood sample. Often, a panel of animals
will have been immunized and the spleen of the animal with the highest
antibody titer will be removed and the spleen lymphocytes obtained by
homogenizing the spleen with a syringe.

[0138]The antibody-producing B lymphocytes from the immunized animal are
then fused with cells of an immortal myeloma cell, generally one of the
same species as the animal that was immunized. Myeloma cell lines suited
for use in hybridoma-producing fusion procedures preferably are
non-antibody-producing, have high fusion efficiency, and enzyme
deficiencies that render them incapable of growing in certain selective
media that support the growth of only the desired fused cells
(hybridomas). Any one of a number of myeloma cells may be used, as are
known to those of skill in the art (e.g., Goding, pp. 65-66, 1986). For
example, where the immunized animal is a mouse, one may use
P3-NS-1-Ag4-1, Sp2/0, P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag41, Sp210-Ag14,
FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may
use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,
LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell
fusions.

[0139]Methods for generating hybrids of antibody-producing spleen or lymph
node cells and myeloma cells usually comprise mixing somatic cells with
myeloma cells in a 2:1 proportion, though the proportion may vary from
about 20:1 to about 1:1, respectively, in the presence of an agent or
agents (chemical or electrical) that promote the fusion of cell
membranes. Fusion methods using Sendai virus have been described by
Kohler and Milstein (1975; 1976), and those using polyethylene glycol
(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of
electrically induced fusion methods is also appropriate (Goding pp.
71-74, 1986).

[0140]Viable, fused hybrids are differentiated from the parental, unfused
cells by culturing in a selective medium. The selective medium generally
contains an agent that blocks the de novo synthesis of nucleotides in the
tissue culture media. Exemplary and preferred agents are aminopterin,
methotrexate, and azaserine. Where aminopterin or methotrexate is used,
the media is supplemented with hypoxanthine and thymidine as a source of
nucleotides (HAT medium). Where azaserine is used, the media is
supplemented with hypoxanthine. A preferred selection medium is HAT. The
only cells that can survive in the selective media are those hybrids
formed from myeloma and B cells.

[0141]This culturing provides a population of hybridomas from which
specific hybridomas are selected. Typically, selection of hybridomas is
performed by culturing the cells by single-clone dilution in microtiter
plates, followed by testing the individual clonal supernatants for the
desired reactivity. The assay should be sensitive, simple and rapid, such
as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque
assays, dot immunobinding assays, and the like. The selected hybridomas
would then be serially diluted and cloned into individual
antibody-producing cell lines, which clones may then be propagated
indefinitely to provide MAbs.

[0142]In accordance with the present invention, fragments of the
monoclonal antibody of the invention may be obtained from the monoclonal
antibody produced as described above, by methods that include digestion
with enzymes such as pepsin or papain and/or cleavage of disulfide bonds
by chemical reduction. Alternatively, monoclonal antibody fragments
encompassed by the present invention may be synthesized using an
automated peptide synthesizer.

[0143]Immunoassay Methods

[0144]Immunocomplex formation. In still further embodiments, the present
invention concerns immunodetection methods for binding, purifying,
removing, quantifying or otherwise generally detecting peptides of
interest. The PDE3 proteins or peptides of the present invention may be
employed to detect antibodies having reactivity therewith or,
alternatively, antibodies prepared in accordance with the present
invention may be employed to detect or purify the PDE3 proteins or
peptides. The steps of various useful immunodetection methods have been
described in the scientific literature, such as, e.g., Nakamura et al.
(1987).

[0145]In general, the immunobinding methods include obtaining a sample
suspected of containing a protein, peptide or antibody, and contacting
the sample with an antibody or protein or peptide in accordance with the
present invention, as the case may be, under conditions effective to
allow the formation of immunocomplexes.

[0146]The detection of immunocomplex formation is well known in the art
and may be achieved through the application of numerous approaches. These
methods are generally based upon the detection of a label or marker, such
as any radioactive, fluorescent, biological or enzymatic tags or labels
of standard use in the art. U.S. Patents concerning the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149 and 4,366,241, each incorporated herein by
reference. One may find additional advantages through the use of a
secondary binding ligand such as a second antibody or a biotin/avidin
ligand binding arrangement, as is known in the art.

[0147]Further methods include the detection of primary immune complexes by
a two-step approach. A second binding ligand, such as an antibody, that
has binding affinity for the target protein, peptide or corresponding
antibody is used to form secondary immune complexes, as described above.
After washing, the secondary immune complexes are contacted with a third
binding ligand or antibody that has binding affinity for the second
antibody, again under conditions effective and for a period of time
sufficient to allow the formation of immune complexes (tertiary immune
complexes). The third ligand or antibody is linked to a detectable label,
allowing detection of the tertiary immune complexes thus formed. This
system may provide for signal amplification if this is desired.

[0148]The immunodetection methods of the present invention may be of
utility in the diagnosis of various disease states. A biological or
clinical sample suspected of containing either the target protein or
peptide or corresponding antibody is used. In certain embodiments,
samples from patients with cardiomyopathy and/or pulmonary hypertension
may be immunoassayed to determine the type and abundance of different
PDE3 isoforms present in one or more tissues. Targeted therapy directed
towards PDE3 may utilize inhibitors and/or activators known to be
selective or specific for one or more PDE3 isoforms that are detected in
the patient's affected tissues.

[0149]Immunohistochemistry. The antibodies of the present invention may be
used in conjunction with fresh-frozen or formalin-fixed,
paraffin-embedded tissue blocks prepared by immunohistochemistry (IHC).
Any H-IC method well known in the art may be used, such as those
described in Diagnostic Immunopathology, 2nd edition, edited by Robert B.
Colvin, Atul K. Bhan and Robert T. McCluskey. Raven Press, New York, 1995
(incorporated herein by reference).

[0150]ELISA. Certain immunoassays are the various types of enzyme-linked
immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the
art. Immunohistochemical detection using tissue sections is also
particularly useful. However, it will be readily appreciated that
detection is not limited to such techniques, and Western blotting, dot
blotting, FACS analyses, and the like may also be used.

[0151]In one exemplary ELISA, antibodies binding to the PDE3 proteins of
the invention are immobilized onto a selected surface exhibiting protein
affinity, such as a well in a polystyrene microtiter plate. Then, a test
composition suspected of containing the PDE3 isoforms, such as a clinical
sample, is added to the wells. After binding and washing to remove
non-specifically bound immunocomplexes, the bound antigen may be
detected. Detection is generally achieved by the addition of a second
antibody specific for the target protein, linked to a detectable label.
This type of ELISA is a simple "sandwich ELISA." Detection may also be
achieved by the addition of a second antibody, followed by the addition
of a third antibody that has binding affinity for the second antibody,
with the third antibody being linked to a detectable label. The skilled
artisan will realize that a variety of ELISA and other immunoassay
techniques are known in the art, any of which may be performed within the
scope of the present invention.

Methods of Immobilization

[0152]In various embodiments, the PDE3 proteins or peptides or anti-PDE3
antibodies of the present invention may be attached to a solid surface
("immobilized"). In a preferred embodiment, immobilization may occur by
attachment to a solid surface, such as a magnetic, glass or plastic bead,
a plastic microtiter plate or a glass slide.

[0153]Immobilization of proteins or peptides may be achieved by a variety
of methods involving either non-covalent or covalent interactions between
the immobilized protein or peptide and an anchor. In an exemplary
embodiment, immobilization may be achieved by coating a solid surface
with a cross-linkable group, such as an amino, carboxyl, sulfhydryl,
alcohol or other group and attaching a protein or peptide using a
cross-linking reagent.

[0154]Homobifunctional reagents that carry two identical functional groups
are highly efficient in inducing cross-linking. Heterobifunctional
reagents contain two different functional groups. By taking advantage of
the differential reactivities of the two different functional groups,
cross-linking can be controlled both selectively and sequentially. The
bifunctional cross-linking reagents can be divided according to the
specificity of their functional groups, e.g., amino, sulfhydryl,
guanidino or carboxyl-specific groups. Of these, reagents directed to
free amino groups have become especially popular because of their
commercial availability, ease of synthesis and the mild reaction
conditions under which they can be applied.

[0155]Exemplary methods for cross-linking molecules are disclosed in U.S.
Pat. Nos. 5,603,872 and 5,401,511. Amine residues may be introduced onto
a surface through the use of aminosilane. Cross-linking reagents include
bisimidates, dinitrobenzene, N-hydroxysuccinimide ester of suberic acid,
disuccinimidyl tartarate, dimethyl-3,3'-dithiobispropionimidate,
N-succinimidyl-3-(2-pyridyldithio)-propionate,
4-(bromoaminoethyl)-2-nitrophenylazide, 4-azidogyloxal and a
water-soluble carbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC). The present invention is not limiting as to the
cross-linking agents that may be used.

Nucleic Acids

[0156]The present invention also provides in another embodiment, genes
encoding PDE3. As discussed below, a "PDE3 gene" may contain a variety of
different bases and yet still produce a corresponding polypeptide that is
indistinguishable functionally, and in some cases structurally, from the
genes disclosed herein. Other embodiments of the invention may concern
nucleic acids (antisense RNAs, ribozymes) that can bind to and inhibit
transcription and/or translation of one or more RNA species encoding a
PDE3A isoform protein. The design and production of antisense RNAs, or
cDNAs encoding antisense RNAs, are well known in the art and any such
known method may be used in the practice of the present invention (e.g.,
U.S. Pat. Nos. 6,210,892; 6,248,724; 6,277,981; 6,300,492; 6,303,374;
6,310,047; 6,365,345). In certain embodiments, an antisense RNA may be
targeted against a particular PDE3A isoform, for example, by selecting a
target sequence that is present in one PDE3A isoform mRNA but not in
another. The term "nucleic acid" encompasses single-stranded,
double-stranded, triple-stranded DNA and/or RNA of any type, as well as
analogs of and chemically modified forms of DNA and/or RNA.

[0157]Any reference to a nucleic acid should be read as encompassing a
host cell containing that nucleic acid and, in some cases, capable of
expressing the product of that nucleic acid. Cells expressing nucleic
acids of the present invention may prove useful in the context of
screening for agents that induce, repress, inhibit, augment, interfere
with, block, abrogate, stimulate, or enhance the catalytic activity,
regulatory properties or subcellular localization of PDE3 isoforms.

[0158]Nucleic Acids Encoding PDE3

[0159]Nucleic acids may contain an entire gene, a cDNA, or a domain of a
PDE3 isoform that expresses catalytic activity, or any other fragment of
the sequences set forth herein. The nucleic acid may be derived from
genomic DNA, i.e., cloned directly from the genome of a particular
organism. In preferred embodiments, however, the nucleic acid would
comprise complementary DNA (cDNA).

[0160]The DNA segments of the present invention include those encoding
biologically functional equivalent PDE3 proteins and peptides. Such
sequences may arise as a consequence of codon redundancy and amino acid
functional equivalency that are known to occur naturally within nucleic
acid sequences and the proteins thus encoded. Alternatively, functionally
equivalent proteins or peptides may be created via the application of
recombinant DNA technology, in which changes in the protein structure may
be engineered, based on considerations of the properties of the amino
acids being exchanged. Changes designed by man may be introduced through
the application of site-directed mutagenesis techniques or may be
introduced randomly and screened later for the desired function, as
described below.

[0161]Assay of PDE3A Isoform mRNA Levels

[0162]Some embodiments of the invention concern methods for determining
the levels of mRNA species encoding the three PDE3A isoforms in various
cells, tissues, organs or other samples. A variety of assays for mRNA
levels are known in the art and any such known assay may be used. The
three PDE3A isoform mRNAs differ in length, not in sequence. Therefore,
any assay for mRNA levels must either separate the mRNAs by size or must
be performed by a subtraction process. The skilled artisan is aware that
RNA species are particularly sensitive to endogenous and/or exogenous
RNAse degradation and that great care must be taken to inhibit or
inactivate RNAse before RNA levels can be determined. Typical procedures
involve treatment of solutions with diethylpyrocarbonate (DEPC) and
autoclaving, as well as addition of commercial RNAse inhibitors.

[0163]Northern blotting is a well-known method for assaying mRNA species
that differ by size. Either total cell RNA or polyadenylated mRNA may be
purified from a sample by known techniques (e.g., Sambrook et al., 1989).
The purified RNA is separated by size using gel electrophoresis. After
transfer to a nylon, nitrocellulose or other membrane, the size-separated
RNAs are probed with a labeled oligonucleotide that hybridizes
specifically with one or more target RNAs. The presence of an RNA species
that hybridizes with the oligonucleotide probe is detected by
autoradiography, fluorography or other known techniques. Further examples
of the use of Northern blotting to detect PDE3A mRNAs are disclosed below
in the Examples section. It appears that in most cell types, a given
PDE3A isoform mRNA will either be present or absent. Thus, generally it
will be sufficient to detect the presence or absence of a PDE3A isoform
mRNA. However, the amounts of each isoform mRNA present in a sample may
also be determined by standard techniques, such as using autoradiography
or fluorography to expose a film (e.g., Kodak X-Omat, Eastman Kodak,
Rochester, N.Y.), and scanning the band intensity on the developed film.

[0164]Other well known methods for detecting and/or quantifying mRNA
species may be used. For example, the target nucleic acids of interest
may be amplified as disclosed below. Amplification products may be
attached to a membrane, 96-well plate, nucleic acid chip or other
substrate and detected. Because the PDE3A isoforms do not differ in
sequence, determination of the amounts of each mRNA species would require
three separate probes. One probe would be designed to be complementary to
the PDE3A3 mRNA sequence and would detect PDE3A1, PDE3A2 and PDE3A3. A
second probe would be designed to be complementary to the 5' portion of
the PDE3A2 mRNA sequence (see SEQ ID NO:15), for example, to the 3' end
of exon 1 or to exons 2 or 3. That probe would hybridize with mRNAs for
PDE3A1 and PDE3A2. A third probe would be designed to be complementary
with the 5' end of exon 1. That probe would only hybridize with the mRNA
encoding PDE3A1 (SEQ ID NO:14, SEQ ID NO:18). By assaying the levels of
PDE3A mRNAs using the three different probes, it would be possible to
determine the amount of each isoform mRNA species by subtraction.

[0165]As discussed in further detail in the Examples section, the PDE3A
isoforms are encoded by at least two, and possibly by three different
mRNAs. PDE3A1 mRNA is translated to a 136 kDa protein isoform, While a
PDE3A2 mRNA may be translated to give both 94 kDA and 118 kDA protein
isoforms. Alternatively, each of the different sized protein isoforms may
be encoded by a separate mRNA species.

[0167]In certain embodiments of the invention, high throughput screening
(HTS) methods directed towards mRNA may be used to assay for inhibitors
and/or activators that affect expression of specific PDE3 isoforms. Such
methods are known in the art and, in some embodiments, may be performed
using kits and/or apparatus obtained from commercial vendors (e.g.,
Xpress-Screen mRNA Detection Assay Service, Applied Biosystems, Foster
City, Calif.). The object of high throughput screening is to survey
thousands of compounds, for example, in the form of small molecule
libraries, phage display libraries, native plant or animal extracts,
combinatorial chemistry libraries, etc., for a pharmaceutically
significant effect on a target protein, cell, tissue, organ or organism.
Effective compounds may be further modified by chemical substitution
and/or modification to provide increased efficacy, safety, duration of
effect, etc.

[0168]HTS assays may be directed against one or more proteins or peptides
of interest, such as PDE3A-136, PDE3A-118, PDE3A-94 or PDE3B-137 using
known techniques. Preferably, libraries of potential inhibitors and/or
activators are exposed to PDE3 proteins and/or peptides and enzyme
catalytic activity and/or regulatory properties are assayed. Such assays
may be performed, for example, in 96-well microtiter plates using known
colorimetric, luminescent and/or radioactive assays for enzyme activity.
In other alternative embodiments, the test peptides and/or proteins may
be attached to a surface, such as a protein chip, microtiter wells,
membrane or other surface known in the art and libraries of compounds may
be screened for their ability to bind to the various PDE3 isoforms.

[0169]Protein-based HTS assays can be laborious and time-consuming. An
alternative method for performing HTS analysis is to screen targets, such
as cells, tissues, organs or organisms, for an effect of a test compound
on mRNA levels. With respect to PDE3 isoforms, such assays may
potentially be directed towards identifying compounds that directly or
indirectly affect PDE3A1 or PDE3A2 mRNA levels. The cell or tissue of
interest, for example, a tissue sample from an individual with dilated
cardiomyopathy or an Sfo cell transfected with a PDE3A-encoding gene, may
be exposed to a series of test compounds in 96- or 384-well microplates.
After incubation and cell lysis, a biotinylated probe specific for the
mRNA of interest is used to hybridize to total cell RNA or to purified
polyadenylated mRNA. The DNA-RNA hybrid may be transferred to a
streptavidin-coated plate, which binds to the biotinylated probe. A
labeled antibody, such as an alkaline phosphatase-conjugated antibody
that binds specifically to RNA-DNA hybrids, is incubated with the plate.
Unbound antibody is removed by washing and the presence of RNA-DNA
hybrids is detected by developing the labeled antibody, for example,
using a chemiluminescent substrate (Xpress-Screen, Applied Biosystems).
In this way, hundreds of test compounds may be screened simultaneously
for an effect on PDE3 isoform expression.

[0170]In alternative embodiments, test compounds may be screened by
looking for secondary effects of PDE3A isoform proteins. Inhibition or
activation of PDE3 activity and/or expression may be determined
indirectly. By affecting the cellular levels of cAMP and/or cGMP, PDE3
isoforms may affect the expression of known cyclic nucleotide-regulated
genes. Cells or tissues that have been exposed to test compounds may be
screened, as described above, for mRNAs encoded by genes that are known
to be dependent on cyclic nucleotide levels. Effects of inhibitors and/or
activators of PDE3 isoforms may be monitored by screening normal,
diseased and/or transformed cells for changes in expression levels of
cAMP- or cGMP-regulated genes.

Nucleic Acid Amplification

[0171]Nucleic acids of use as a template for amplification may be isolated
from cells contained in a biological sample according to standard
methodologies (Sambrook et al., 1989). The nucleic acid may be genomic
DNA or fractionated or whole cell RNA. Where RNA is used, it may be
desired to convert the RNA to a complementary cDNA. In one embodiment,
the RNA is whole-cell RNA and is used directly as the template for
amplification. In other embodiments, the RNA may be polyadenylated mRNA.
Purification of mRNA, for example, by affinity chromatography to oligo-dT
columns, is well known in the art.

[0172]Pairs of primers that selectively hybridize to nucleic acids
corresponding to specific markers are contacted with the isolated nucleic
acid under conditions that permit selective hybridization. Once
hybridized, the nucleic acid:primer complex is contacted with one or more
enzymes that facilitate template-dependent nucleic acid synthesis.
Multiple rounds of amplification, also referred to as "cycles," are
conducted until a sufficient amount of amplification product is produced.

[0173]Next, the amplification product is detected. In certain
applications, the detection may be performed by visual means.
Alternatively, the detection may involve indirect identification of the
product via chemiluminescence, radioactive scintigraphy of incorporated
radiolabel or fluorescent label or even via a system using electrical or
thermal impulse signals (Affymax Technology; Bellus, 1994).

[0174]Following detection, one may compare the results seen in a given
patient with a statistically significant reference group of normal
patients and patients exhibiting a disease state. In this way, it is
possible to correlate the amount of marker detected with various clinical
states.

[0175]Primers

[0176]The term "primer," as defined herein, is meant to encompass any
nucleic acid that is capable of priming the synthesis of a nascent
nucleic acid in a template-dependent process. Typically, primers are
oligonucleotides from ten to twenty base pairs in length, but longer
sequences may be employed. Primers may be provided in double-stranded or
single-stranded form, although the single-stranded form is preferred.

[0177]Template-Dependent Amplification Methods

[0178]A number of template-dependent processes are available to amplify
the marker sequences present in a given template sample. One of the best
known amplification methods is the polymerase chain reaction (referred to
as PCR), which is described in detail in U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,800,159, and in Innis et al., 1990, each of which is
incorporated herein by reference in its entirety.

[0179]A reverse transcriptase PCR amplification procedure may be performed
in order to quantify the amount of mRNA amplified. Methods of reverse
transcribing RNA into cDNA are well known and described in Sambrook et
al., 1989. Alternative methods for reverse transcription utilize
thermostable DNA polymerases. These methods are described in WO 90/07641
filed Dec. 21, 1990. Polymerase chain reaction methodologies are well
known in the art.

[0180]Another method for amplification is the ligase chain reaction
("LCR"), disclosed in European Application No. 320,308, incorporated
herein by reference in its entirety. In LCR, two complementary probe
pairs are prepared, and in the presence of the target sequence, each pair
will bind to opposite complementary strands of the target such that they
abut. In the presence of a ligase, the two probe pairs will link to form
a single unit. By temperature cycling, as in PCR, bound ligated units
dissociate from the target and then serve as "target sequences" for
ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a
method similar to LCR for binding probe pairs to a target sequence.

[0181]Qbeta Replicase, described in PCT Application No. PCT/US87/00880,
may also be used as still another amplification method in the present
invention. In this method, a replicative sequence of RNA that has a
region complementary to that of a target is added to a sample in the
presence of an RNA polymerase. The polymerase will copy the replicative
sequence that may then be detected.

[0182]An isothermal amplification method in which restriction
endonucleases and ligases are used to achieve the amplification of target
molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one
strand of a restriction site may also be useful in the amplification of
nucleic acids in the present invention. Walker et al., Proc. Nat'l Acad.
Sci. USA 89:392-396 (1992), incorporated herein by reference in its
entirety.

[0183]Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acids that involves
multiple rounds of strand displacement and synthesis, i.e., nick
translation. A similar method, called Repair Chain Reaction (RCR),
involves annealing several probes throughout a region targeted for
amplification, followed by a repair reaction in which only two of the
four bases are present. The other two bases may be added as biotinylated
derivatives for easy detection. A similar approach is used in SDA.
Target-specific sequences may also be detected using a cyclic probe
reaction (CPR). In CPR, a probe having 3' and 5' sequences of
non-specific DNA and a middle sequence of specific RNA is hybridized to
DNA that is present in a sample. Upon hybridization, the reaction is
treated with RNase H, and the products of the probe identified as
distinctive products that are released after digestion. The original
template is annealed to another cycling probe and the reaction is
repeated.

[0184]Still other amplification methods described in GB Application No. 2
202 328 and in PCT Application No. PCT/US89/01025, each of which is
incorporated herein by reference in its entirety, may be used in
accordance with the present invention. In the former application,
"modified" primers are used in a PCR-like template and enzyme-dependent
synthesis. The primers may be modified by labeling with a capture moiety
(e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter
application, an excess of labeled probes are added to a sample. In the
presence of the target sequence, the probe binds and is cleaved
catalytically. After cleavage, the target sequence is released intact to
be bound by excess probe. Cleavage of the labeled probe signals the
presence of the target sequence.

[0185]Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic acid
sequence-based amplification (NASBA) and 3SR (Kwoh et al., Proc. Nat'l
Acad. Sci. USA 86:1173 (1989); Gingeras et al., PCT Application WO
88/10315, incorporated herein by reference in their entirety). In NASBA,
the nucleic acids may be prepared for amplification by standard
phenol/chloroform extraction, heat denaturation of a clinical sample,
treatment with lysis buffer and minispin columns for isolation of DNA and
RNA, or guanidinium chloride extraction of RNA. These amplification
techniques involve annealing a primer that has target-specific sequences.
Following polymerization, DNA/RNA hybrids are digested with RNase H,
while double-stranded DNA molecules are heat denatured again. In either
case, the single-stranded DNA is made fully double-stranded by addition
of a second target-specific primer, followed by polymerization. The
double-stranded DNA molecules are then multiply transcribed by a
polymerase, such as T7 or SP6. In an isothermal cyclic reaction, the RNAs
are reverse transcribed into double-stranded DNA, and transcribed once
again with a polymerase, such as T7 or SP6. The resulting products,
whether truncated or complete, indicate target-specific sequences.

[0186]Davey et al., European Application No. 329 822 (incorporated herein
by reference in its entirety), disclose a nucleic acid amplification
process involving cyclically synthesizing single-stranded RNA ("ssRNA"),
single-stranded DNA ("ssDNA"), and double-stranded DNA ("dsDNA"), which
may be used in accordance with the present invention. The ssRNA is a
first template for a first primer oligonucleotide, which is elongated by
reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then
removed from the resulting DNA:RNA duplex by the action of ribonuclease H
(RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
The resultant ssDNA is a second template for a second primer, which also
includes the sequences of an RNA polymerase promoter (exemplified by T7
RNA polymerase) 5' to its homology to the template. This primer is then
extended by DNA polymerase (exemplified by the large "Klenow" fragment of
E. coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA between
the primers and having additionally, at one end, a promoter sequence.
This promoter sequence may be used by the appropriate RNA polymerase to
make many RNA copies of the DNA. These copies may then re-enter the cycle
leading to very swift amplification. With proper choice of enzymes, this
amplification may be done isothermally without addition of enzymes at
each cycle. Because of the cyclical nature of this process, the starting
sequence may be chosen to be in the form of either DNA or RNA.

[0187]Miller et al., PCT Application WO 89/06700 (incorporated herein by
reference in its entirety), disclose a nucleic acid sequence
amplification scheme based on the hybridization of a promoter/primer
sequence to a target single-stranded DNA ("ssDNA"), followed by
transcription of many RNA copies of the sequence. This scheme is not
cyclic, i.e., new templates are not produced from the resultant RNA
transcripts. Other amplification methods include "race" and "one-sided
PCR" (M. A. Frohman, in PCR PROTOCOLS: A GUIDE TO METHODS AND
APPLICATIONS, Academic Press, N.Y. (1990), and Ohara et al., Proc. Nat'l
Acad. Sci. USA, 86:5673-5677 (1989), each herein incorporated by
reference in their entirety).

[0188]Methods based on ligation of two (or more) oligonucleotides in the
presence of nucleic acid having the sequence of the resulting
"di-oligonucleotide," thereby amplifying the di-oligonucleotide, may also
be used in the amplification step of the present invention (Wu et al.,
Genomics 4:560 (1989), incorporated herein by reference in its entirety).

[0189]Separation Methods

[0190]Following amplification, it may be desirable to separate the
amplification product from the template and the excess primer for the
purpose of determining whether specific amplification has occurred. In
one embodiment, amplification products are separated by agarose,
agarose-acrylamide or polyacrylamide gel electrophoresis using standard
methods. See Sambrook et al., 1989.

[0191]Alternatively, chromatographic techniques may be employed to effect
separation. There are many kinds of chromatography that may be used in
the present invention: adsorption, partition, ion-exchange and molecular
sieve, and many specialized techniques for using them including column,
paper, thin-layer and gas chromatography (Freifelder, 1982).

[0192]Identification Methods

[0193]Amplification products must be visualized in order to confirm
amplification of the marker sequences. One typical visualization method
involves staining of a gel with ethidium bromide and visualization under
UV light. Alternatively, if the amplification products are integrally
labeled with radio- or fluorometrically-labeled nucleotides, the
amplification products may then be exposed to x-ray film or visualized
under the appropriate stimulating spectra, following separation.

[0194]In one embodiment, visualization is achieved indirectly. Following
separation of amplification products, a labeled, nucleic acid probe is
brought into contact with the amplified marker sequence. The probe
preferably is conjugated to a chromophore but may be radiolabeled. In
another embodiment, the probe is conjugated to a binding partner, such as
an antibody or biotin, where the other member of the binding pair carries
a detectable moiety.

[0195]In one embodiment, detection is by Southern blotting and
hybridization with a labeled probe. The techniques involved in Southern
blotting are well known to those of skill in the art and may be found in
many standard books on molecular protocols. See Sambrook et al., 1989.
Briefly, amplification products are separated by gel electrophoresis. The
gel is then contacted with a membrane, such as nitrocellulose, permitting
transfer of the nucleic acid and non-covalent binding. Subsequently, the
membrane is incubated with a chromophore-conjugated probe that is capable
of hybridizing with a target amplification product. Detection is by
exposure of the membrane to x-ray film or ion-emitting detection devices.

[0196]One example of the foregoing is described in U.S. Pat. No.
5,279,721, incorporated by reference herein, which discloses an apparatus
and method for the automated electrophoresis and transfer of nucleic
acids. The apparatus permits electrophoresis and blotting without
external manipulation of the gel and is ideally suited to carrying out
methods according to the present invention.

Antisense Constructs, Ribozymes and Small Interfering RNAs

[0197]Antisense

[0198]The term "antisense" refers to polynucleotide molecules
complementary to a portion of a targeted gene or mRNA species.
Complementary polynucleotides are those that are capable of base-pairing
according to the standard Watson-Crick complementarity rules. That is,
purines will base pair with pyrimidines to form combinations of guanine
paired with cytosine (G:C) and adenine paired with either thymine (A:T)
in the case of DNA, or adenine paired with uracil (A:U) in the case of
RNA. Inclusion of less common bases such as inosine, 5-methylcytosine,
6-methyladenine, hypoxanthine and others in hybridizing sequences does
not interfere with pairing.

[0199]Antisense polynucleotides, when introduced into a target cell,
specifically bind to their target polynucleotide and interfere with
transcription, RNA processing, transport, translation and/or stability.
Antisense RNA constructs, or DNA encoding such antisense RNAs, may be
employed to inhibit gene transcription or translation or both within a
host cell, either in vitro or I, such as within a host animal, including
a human subject.

[0200]The intracellular concentration of monovalent cations is
approximately 160 mM (10 mM Na+; 150 mM K+). The intracellular
concentration of divalent cations is approximately 20 mM (18 mM Mg+;
2 mM Ca++). The intracellular protein concentration, which would
serve to decrease the volume of hybridization and, therefore, increase
the effective concentration of nucleic acid species, is 150 mg/ml.
Constructs may be tested for specific hybridization in vitro under
conditions that mimic these in vivo conditions.

[0201]Antisense constructs may be designed to bind to the promoter and
other control' regions, exons, introns or even exon-intron boundaries of
a gene. In certain embodiments, it is contemplated that effective
antisense constructs may include regions complementary to the mRNA start
site. In preferred embodiments, the antisense constructs are targeted to
a sequence of an hnRNA and/or mRNA that is present in one PDE3A isoform
and not in another. For example, one might target the 5' end of the mRNA
encoding PDE3A1 (SEQ ID NO:14, SEQ ID NO:18), which is missing in the
PDE3A2 mRNA (SEQ ID NO:15). One of ordinary skill in the art can readily
test such constructs to determine whether levels of the target protein
are affected.

[0202]As used herein, the terms "complementary" or "antisense" mean
polynucleotides that are substantially complementary to the target
sequence over their entire length and have very few base mismatches. For
example, sequences of fifteen bases in length may be termed complementary
when they have a complementary nucleotide at thirteen or fourteen
nucleotides out of fifteen. Naturally, sequences that are "completely
complementary" will be sequences that are entirely complementary
throughout their entire length and have no base mismatches.

[0203]Other sequences with lower degrees of homology also are
contemplated. For example, an antisense construct that has limited
regions of high homology, but also contains a non-homologous region
(e.g., a ribozyme) could be designed. These molecules, though having less
than 50% homology, would bind to target sequences under appropriate
conditions.

[0204]Although the antisense sequences may be full-length cDNA copies, or
large fragments thereof, they also may be shorter fragments, or
"oligonucleotides," defined herein as polynucleotides of 50 or less
bases. Although shorter oligomers (8 to 20) are easier to make and
increase in vivo accessibility, numerous other factors are involved in
determining the specificity of base-pairing. For example, both binding
affinity and sequence specificity of an oligonucleotide to its
complementary target increase with increasing length. It is contemplated
that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30, 35, 40, 45, 50 or 100 base pairs will be used. While all or
part of the gene sequence may be employed in the context of antisense
construction, statistically, any sequence of 14 bases long should occur
only once in the human genome and, therefore, suffice to specify a unique
target sequence.

[0205]In certain embodiments, one may wish to employ antisense constructs
that include other elements, for example, those that include C-5 propyne
pyrimidines. Oligonucleotides that contain C-5 propyne analogues of
uridine and cytidine have been shown to bind RNA with high affinity and
to be potent antisense inhibitors of gene expression (Wagner et al.,
1993).

[0206]Alternatively, the antisense oligo- and polynucleotides according to
the present invention may be provided as RNA via transcription from
expression constructs that carry nucleic acids encoding the oligo- or
polynucleotides. Throughout this application, the term "expression
construct" is meant to include any type of genetic construct containing a
nucleic acid encoding a product in which part or all of the nucleic acid
sequence is capable of being transcribed. Typical expression vectors
include bacterial plasmids or phage, such as any of the pUC or
Bluescript® plasmid series or, as discussed further below, viral
vectors adapted for use in eukaryotic cells.

[0207]In preferred embodiments, the nucleic acid encodes an antisense
oligo- or polynucleotide under transcriptional control of a promoter. A
"promoter" refers to a DNA sequence recognized by an RNA polymerase to
initiate the specific transcription of a gene. The phrase "under
transcriptional control" means that the promoter is in the correct
location and orientation in relation to the nucleic acid to control RNA
polymerase initiation.

[0208]The term promoter will be used herein to refer to a group of
transcriptional control modules that are clustered around the initiation
site for RNA polymerase II. Promoters are composed of discrete functional
modules, each consisting of approximately 7 to 20 by of DNA, and
containing one or more recognition sites for transcriptional activator or
repressor proteins. At least one module in each promoter functions to
position the start site for RNA synthesis. The best known example of this
is the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase gene and
the promoter for the SV40 late genes, a discrete element overlying the
start site itself helps to fix the place of initiation.

[0209]Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the region 30
to 110 by upstream of the start site, although a number of promoters have
recently been shown to contain functional elements downstream of the
start site as well. The spacing between promoter elements frequently is
flexible, so that promoter function is preserved when elements are
inverted or moved relative to one another. In the tk promoter, the
spacing between promoter elements can be increased to 50 by apart before
activity begins to decline. Depending on the promoter, it appears that
individual elements can function either co-operatively or independently
to activate transcription.

[0210]The particular promoter that is employed to control the expression
of a nucleic acid encoding the inhibitory polynucleotide is not believed
to be important, so long as it is capable of expressing the peptide in
the targeted cell. Thus, where a human cell is targeted, it is preferable
to position the nucleic acid coding the inhibitory peptide adjacent to
and under the control of a promoter that is active in the human cell.
Generally speaking, such a promoter might include either a human or viral
promoter.

[0211]In various embodiments, the human cytomegalovirus (CMV) immediate
early gene promoter, the SV40 early promoter and the Rous sarcoma virus
long terminal repeat can be used to obtain high-level transcription. The
use of other viral or mammalian cellular or bacterial phage promoters
that are well-known in the art is contemplated as well, provided that the
levels of transcription and/or translation are sufficient for a given
purpose.

[0212]Selection of a promoter that is regulated in response to specific
physiologic signals can permit inducible expression of an antisense
sequence. For example, a nucleic acid under control of the human PAI-1
promoter results in expression inducible by tumor necrosis factor.
Additionally, any promoter/enhancer combination also could be used to
drive expression of a nucleic acid according to the present invention.
Tables 1 and 2 list elements/promoters that may be employed to regulate
transcription and/or translation of operably coupled genes. This list is
exemplary only and any known promoter and/or regulatory element may be
used.

[0214]Another method for inhibiting the expression of specific PDE3A
isoforms is via ribozymes. Ribozymes are RNA-protein complexes that
cleave nucleic acids in a site-specific fashion. Ribozymes have specific
catalytic domains that possess endonuclease activity (Kim and Cech,
1987). For example, a large number of ribozymes accelerate phosphoester
transfer reactions with a high degree of specificity, often cleaving only
one of several phosphoesters in an oligonucleotide substrate (Cech et at,
1981). This specificity has been attributed to the requirement that the
substrate bind via specific base-pairing interactions to an internal
guide sequence ("IGS") of the ribozyme prior to chemical reaction.

[0215]Ribozyme catalysis has primarily been observed as part of
sequence-specific cleavage/ligation reactions involving nucleic acids
(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855
reports that certain ribozymes can act as endonucleases with a sequence
specificity greater than that of known ribonucleases. Thus,
sequence-specific ribozyme-mediated inhibition of gene expression may be
particularly suited to therapeutic applications (Scanlon et al., 1991;
Sarver et al., 1990; Sioud et al., 1992). It was reported that ribozymes
elicited genetic changes in some cell lines to which they were applied.
The altered genes included the oncogenes H-ras, c-fos and genes of HIV.

[0217]The other variable in ribozyme design is the selection of a cleavage
site on a given target RNA. Ribozymes are targeted to a given sequence by
virtue of annealing to a site by complimentary base pair interactions.
Two stretches of homology are required for this targeting. These
stretches of homologous sequences flank the catalytic ribozyme structure
defined above. Each stretch of homologous sequence can vary in length
from 7 to 15 nucleotides. The only requirement for defining the
homologous sequences is that, on the target RNA, they are separated by a
specific sequence that is the cleavage site. For hammerhead ribozymes,
the cleavage site is a dinucleotide sequence on the target RNA, a uracil
(U) followed by either an adenine, cytosine or uracil (A, C or U)
(Perriman et al., 1992).

[0218]The large number of possible cleavage sites in genes of moderate
size, coupled with the growing number of sequences with demonstrated
catalytic RNA cleavage activity, indicates that a large number of
ribozymes that have the potential to down-regulate gene expression are
available. Additionally, due to the sequence variation among different
genes, ribozymes could be designed to specifically cleave individual
genes or gene products. Designing and testing ribozymes for efficient
cleavage of a target RNA is a process well known to those skilled in the
art. Examples of scientific methods for designing and testing ribozymes
are described by Chowrira et al., (1994), incorporated herein by
reference.

[0219]Small Interfering mRNAs

[0220]Another possibility is to inhibit the translation of individual PDE3
mRNAs by RNA interference. This method of post-transcriptional gene
silencing involves the use of a 21- or 22-nucleotide double-stranded
synthetic RNA molecule homologous to a unique nucleotide sequence in the
mRNA of interest. Through a mechanism yet to be determined, such small
interfering RNA molecules (siRNAs) have the ability to reduce expression
of the cognate protein. This approach has been used to reduce the
expression of several cytoskeletal proteins. As noted above, a unique
sequence in PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18) has been identified
that may allow specific interference with the expression of PDE3A-136.

[0221]Methods for selectively interfering with gene expression using small
interfering RNA species ("siRNA") are known in the art (e.g., Bass, 2001;
Elbashir et al., 2001). Short, double-stranded RNAs (dsRNA) of about 30
by or less that are homologous in sequence to a gene to be silenced
(e.g., PDE3A) are introduced into a target cell (Elbashir et al., 2001).
By a poorly understood endogenous pathway, the dsRNAs are broken into
smaller fragments of about 21 to 22 by (siRNAs). These fragments trigger
the degradation of homologous mRNA sequences (Elbashir et al., 2001),
e.g., PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18). Use of siRNAs can
decrease expression of a target gene or even eliminate it entirely (Bass,
2001). Another advantage of siRNAs is that they are effective at lower
concentrations (about 1 to 25 nM) than antisense constructs (Bass, 2001;
Elbashir et al., 2001).

[0222]Transfection of 21 by dsRNA sequences into NIH/3T3 cells, COS-7
cells and Hela S3 cells using cationic liposomes resulted in inhibition
of homologous reporter genes (Elbashir et al., 2001). The effectiveness
of inhibition appeared to be inversely related to the expression levels
of the target gene, with highly expressed genes showing less inhibition
by siRNA constructs (Elbashir et al., 2001). Because the PDE3 genes are
expressed at relatively low levels compared to highly expressed mammalian
genes, the use of siRNA inhibitors should prove effective at inhibiting
or eliminating expression of targeted PDE3 isoforms.

[0223]Expression Vectors

[0224]Nucleic acids encoding PDE3 isoform proteins or peptides may be
incorporated into expression vectors for production of the encoded
proteins or peptides. Non-limiting examples of expression systems known
in the art include bacteria such as E. coli, yeast such as Pichia
pastoris, baculovirus, and mammalian expression systems such as in COS or
CHO cells. A complete gene can be expressed or, alternatively, fragments
of the gene encoding portions of polypeptide can be produced.

[0225]The gene or gene fragment encoding a polypeptide may be inserted
into an expression vector by standard subcloning techniques. An E. coli
expression vector may be used that produces the recombinant polypeptide
as a fusion protein, allowing rapid affinity purification of the protein.
Examples of such fusion protein expression systems are the glutathione
S-transferase system (Pharmacia, Piscataway, N.J.), the maltose binding
protein system (NEB, Beverley, Mass.), the FLAG system (IBI, New Haven,
Conn.), and the 6×His system (Qiagen, Chatsworth, Calif.).

[0226]Some of these systems produce recombinant polypeptides bearing only
a small number of additional amino acids, which are unlikely to affect
the antigenic ability of the recombinant polypeptide. For example, both
the FLAG system and the 6xHis system add only short sequences, both of
which are known to be poorly antigenic and which do not adversely affect
folding of the polypeptide to its native conformation. Other fusion
systems are designed to produce fusions wherein the fusion partner is
easily excised from the desired polypeptide. In one embodiment, the
fusion partner is linked to the recombinant polypeptide by a peptide
sequence containing a specific recognition sequence for a protease.
Examples of suitable sequences are those recognized by the Tobacco Etch
Virus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa (New
England Biolabs, Beverley, Mass.).

[0227]The expression system used may also be one driven by the baculovirus
polyhedron promoter. The gene encoding the polypeptide may be manipulated
by standard techniques in order to facilitate cloning into the
baculovirus vector. One baculovirus vector is the pBlueBac vector
(Invitrogen, Sorrento, Calif.). The vector carrying the gene for the
polypeptide is transfected into Spodoptera frugiperda (Sf9) cells by
standard protocols, and the cells are cultured and processed to produce
the recombinant antigen. See U.S. Pat. No. 4,215,051 (incorporated herein
by reference).

[0228]Amino acid sequence variants of the polypeptide may also be
prepared. These may, for instance, be minor sequence variants of the
polypeptide that arise due to natural variation within the population or
they may be homologues found in other species. They also may be sequences
that do not occur naturally but are sufficiently similar so that they
function similarly and/or elicit an immune response that cross-reacts
with natural forms of the polypeptide. Sequence variants may be prepared
by standard methods of site-directed mutagenesis such as those described
herein.

[0229]Substitutional variants typically contain an alternative amino acid
at one or more sites within the protein, and may be designed to modulate
one or more properties of the polypeptide such as stability against
proteolytic cleavage. Substitutions preferably are conservative, that is,
one amino acid is replaced with one of similar size and charge.
Conservative substitutions are well known in the art and include, for
example, the changes of: arginine to lysine; asparagine to glutamine or
histidine; aspartate to glutamate; cysteine to serine; glutamine to
asparagine; glutamate to aspartate; histidine to asparagine or glutamine;
isoleucine to leucine or valine; leucine to valine or isoleucine; lysine
to arginine or glutamine; methionine to leucine or isoleucine;
phenylalanine to tyrosine; serine to threonine; tyrosine to tryptophan or
phenylalanine; and valine to isoleucine or leucine.

[0230]In making such changes, the hydropathic index of amino acids may be
considered. The importance of the hydropathic amino acid index in
conferring interactive biologic function on a protein is generally
understood in the art (Kyte and Doolittle, 1982). It is accepted that the
relative hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines the
interaction of the protein with other molecules. Each amino acid has been
assigned a hydropathic index on the basis of its hydrophobicity and
charge characteristics (Kyte and Doolittle, 1982); these are: Isoleucine
(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine
(-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine
(-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).

[0231]It is known in the art that certain amino acids may be substituted
by other amino acids having a similar hydropathic index or score and
still result in a protein with similar biological activity, i.e., still
obtain a biological functionally equivalent protein. In making such
changes, the substitution of amino acids whose hydropathic indices are
within ±2 is preferred, those that are within ±1 are particularly
preferred, and those within ±0.5 are even more particularly preferred.

[0232]It is also understood in the art that the substitution of like amino
acids can be made effectively on the basis of hydrophilicity (U.S. Pat.
No. 4,554,101, incorporated herein by reference). The following
hydrophilicity values have been assigned to amino acid residues: arginine
(+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);
serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);
threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5);
cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan
(-3.4). It is understood that an amino acid can be substituted for
another having a similar hydrophilicity value and still obtain a
biologically equivalent and immunologically equivalent protein. In such
changes, the substitution of amino acids whose hydrophilicity values are
within ±2 is preferred, those that are within ±1 are particularly
preferred, and those within ±0.5 are even more particularly preferred.

[0233]Insertional variants include fusion proteins such as those used to
allow rapid purification of the polypeptide and also may include hybrid
proteins containing sequences from other proteins and polypeptides that
are homologues of the polypeptide. For example, an insertional variant
may include portions of the amino acid sequence of the polypeptide from
one species, together with portions of the homologous polypeptide from
another species. Other insertional variants may include those in which
additional amino acids are introduced within the coding sequence of the
polypeptide. These typically are smaller insertions than the fusion
proteins described above and are introduced, for example, to disrupt a
protease cleavage site.

[0234]The engineering of DNA segment(s) for expression in a prokaryotic or
eukaryotic system may be performed by techniques generally known to those
of skill in recombinant expression. It is believed that virtually any
expression system may be employed in the expression of the claimed
nucleic acid sequences.

[0235]As used herein, the terms "engineered" and "recombinant" cells are
intended to refer to a cell into which an exogenous DNA segment or gene,
such as a cDNA or gene has been introduced through the hand of man.
Therefore, engineered cells are distinguishable from naturally occurring
cells that do not contain a recombinantly introduced exogenous DNA
segment or gene. Recombinant cells include those having an introduced
cDNA or genomic gene, and also include genes positioned adjacent to a
heterologous promoter not naturally associated with the particular
introduced gene.

[0236]To express a recombinant encoded protein or peptide, whether mutant
or wild-type, in accordance with the present invention, one would prepare
an expression vector that comprises one of the claimed isolated nucleic
acids under the control of, or operatively linked to, one or more
promoters. To bring a coding sequence "under the control of a promoter,
one positions the 5' end of the transcription initiation site of the
transcriptional reading frame generally between about 1 and about 50
nucleotides "downstream" (i.e., 3') of the chosen promoter. The
"upstream" promoter stimulates transcription of the DNA and promotes
expression of the encoded recombinant protein. This is the meaning of
"recombinant expression" in this context.

[0237]Many standard techniques are available to construct expression
vectors containing the appropriate nucleic acids and
transcriptional/translational control sequences in order to achieve
protein or peptide expression in a variety of host-expression systems.
Cell types available for expression include, but are not limited to,
bacteria, such as E. coli and B. subtilis transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors.

[0238]Promoters that are most commonly used in recombinant DNA
construction include the β-lactamase (penicillinase), lactose and
tryptophan (trp) promoter systems. While these are the most commonly
used, other microbial promoters have been discovered and utilized, and
details concerning their nucleotide sequences have been published,
enabling those of skill in the art to ligate them functionally with
plasmid vectors.

[0239]For expression in Saccharomyces, the plasmid YRp7, for example, is
commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper
et al., 1980). This plasmid already contains the trp1 gene that provides
a selection marker for a mutant strain of yeast lacking the ability to
grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. The presence
of the trp1 lesion as a characteristic of the yeast host cell genome then
provides an effective environment for detecting transformation by growth
in the absence of tryptophan.

[0242]In addition to microorganisms, cultures of cells derived from
multicellular organisms may also be used as hosts. In principle, any such
cell culture is workable, whether from vertebrate or invertebrate
culture. In addition to mammalian cells, these include insect cell
systems infected with recombinant virus expression vectors (e.g.,
baculovirus); and plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or transformed with recombinant plasmid expression vectors
(e.g., Ti plasmid) containing one or more coding sequences.

[0243]In a useful insect system, Autographa californica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express foreign genes.
The virus grows in Spodoptera frugiperda cells. The isolated nucleic acid
coding sequences are cloned into non-essential regions (for example, the
polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example, the polyhedrin promoter). Successful insertion of
the coding sequences results in the inactivation of the polyhedrin gene
and production of non-occluded recombinant virus (i.e., virus lacking the
protein coat coded for by the polyhedrin gene). These recombinant viruses
are then used to infect Spodoptera frugiperda cells in which the inserted
gene is expressed (e.g., U.S. Pat. No. 4,215,051 (Smith)).

[0244]Examples of useful mammalian host cell lines are VERO and HeLa
cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293,
HepG2, 3T3, RIN and MDCK cell lines. In addition, a host cell strain may
be chosen that modulates the expression of the inserted sequences, or
modifies and processes the gene product in the specific fashion desired.
Such modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the encoded
protein.

[0245]Different host cells have characteristic and specific mechanisms for
the post-translational processing and modification of proteins.
Appropriate cell lines or host systems may be chosen to ensure the
correct modification and processing of the foreign protein expressed.
Expression vectors for use in mammalian cells ordinarily include an
origin of replication (as necessary), a promoter located in front of the
gene to be expressed, along with any necessary ribosome binding sites,
RNA splice sites, polyadenylation site, and transcriptional terminator
sequences. The origin of replication may be provided either by
construction of the vector to include an exogenous origin, such as may be
derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source,
or may be provided by the host cell chromosomal replication mechanism. If
the vector is integrated into the host cell chromosome, the latter is
often sufficient.

[0246]The promoters may be derived from the genome of mammalian cells
(e.g., metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter, the vaccinia virus 7.5K promoter). Further, it
is also possible, and may be desirable, to utilize promoter or control
sequences normally associated with the desired gene sequence, provided
such control sequences are compatible with the host cell systems.

[0247]A number of viral-based expression systems may be utilized; for
example, commonly used promoters are derived from polyoma, Adenovirus 2,
and most frequently Simian Virus 40 (SV40). The early and late promoters
of SV40 virus are particularly useful because both are obtained easily
from the virus as a fragment that also contains the SV40 viral origin of
replication. Smaller or larger SV40 fragments may also be used, provided
there is included the approximately 250 by sequence extending from the
HindIII site toward the BglI site located in the viral origin of
replication.

[0248]In cases where an adenovirus is used as an expression vector, the
coding sequences may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter and
tripartite leader sequence. This chimeric gene may then be inserted in
the adenovirus genome by in vitro or in vivo recombination. Insertion in
a non-essential region of the viral genome (e.g., region E1 or E3) will
result in a recombinant virus that is viable and capable of expressing
proteins in infected hosts.

[0249]Specific initiation signals may also be required for efficient
translation of the claimed isolated nucleic acid coding sequences. These
signals include the ATG initiation codon and adjacent sequences.
Exogenous translational control signals, including the ATG initiation
codon, may additionally need to be provided. One of ordinary skill in the
art would readily be capable of determining this and providing the
necessary signals. It is well known that the initiation codon must be
in-frame (or in-phase) with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These exogenous
translational control signals and initiation codons may be of a variety
of origins, both natural and synthetic. The efficiency of expression may
be enhanced by the inclusion of appropriate transcription enhancer
elements or transcription terminators (Bittner et al., 1987).

[0250]In eukaryotic expression, one will also typically desire to
incorporate into the transcriptional unit an appropriate polyadenylation
site (e.g., 5'-AATAAA-3') if one was not contained within the original
cloned segment. Typically, the poly A addition site is placed about 30 to
2000 nucleotides "downstream" of the termination site of the protein at a
position prior to transcription termination.

[0251]For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines that stably express
constructs encoding proteins may be engineered. Rather than using
expression vectors that contain viral origins of replication, host cells
may be transformed with vectors controlled by appropriate expression
control elements (e.g., promoter, enhancer, sequences, transcription
terminators, polyadenylation sites, etc.), and a selectable marker.
Following the introduction of foreign DNA, engineered cells may be
allowed to grow for one to two days in an enriched media, and then are
switched to a selective media. The selectable marker in the recombinant
plasmid confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form foci, which
in turn may be cloned and expanded into cell lines.

[0253]Site-specific mutagenesis is a technique useful in the preparation
of individual peptides, or biologically functional equivalent proteins or
peptides, through specific mutagenesis of the underlying DNA. The
technique further provides a ready ability to prepare and test sequence
variants, incorporating one or more of the foregoing considerations, by
introducing one or more nucleotide sequence changes into the DNA.
Site-specific mutagenesis allows the production of mutants through the
use of specific oligonucleotide sequences that encode the DNA sequence of
the desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and sequence
complexity to form a stable duplex on both sides of the deletion junction
being traversed. Typically, a primer of about 17 to 25 nucleotides in
length is preferred, with about 5 to 10 residues on both sides of the
junction of the sequence being altered.

[0254]In general, the technique of site-specific mutagenesis is well known
in the art. As will be appreciated, the technique typically employs a
bacteriophage vector that exists in both a single-stranded and
double-stranded form. Typical vectors useful in site-directed mutagenesis
include vectors such as the M13 phage. These phage vectors are
commercially available and their use is generally well known to those
skilled in the art. Double-stranded plasmids are also routinely employed
in site-directed mutagenesis, which eliminates the step of transferring
the gene of interest from a phage to a plasmid.

[0255]In general, site-directed mutagenesis is performed by first
obtaining a single-stranded vector, or melting of two strands of a
double-stranded vector that includes within its sequence a DNA sequence
encoding the desired protein. An oligonucleotide primer bearing the
desired mutated sequence is synthetically prepared. This primer is then
annealed with the single-stranded DNA preparation and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment, in
order to complete the synthesis of the mutation-bearing strand. Thus, a
heteroduplex is formed wherein one strand encodes the original
non-mutated sequence and the second strand bears the desired mutation.
This heteroduplex vector is then used to transform appropriate cells,
such as E. coli cells, and clones are selected that include recombinant
vectors bearing the mutated sequence arrangement.

Phage Display

[0256]In certain embodiments, it may be desirable to use random amino acid
sequences in the form of a phage display library for use as potential
isoform-selective PDE3 inhibitors or activators. The phage display method
has been used for a variety of purposes (see, for example, Scott and
Smith, 1990, 1993; U.S. Pat. Nos. 5,565,332, 5,596,079, 6,031,071 and
6,068,829, each incorporated herein by reference).

[0257]Generally, a phage display library is prepared by first constructing
a partially randomized library of cDNA sequences, encoding a large number
of amino acid combinations. The cDNA sequences are inserted in frame
into, for example, a viral coat protein for a phage such as the fuse 5
vector (U.S. Pat. No. 6,068,829). The cDNAs are expressed as random amino
acid sequences, incorporated into a coat protein. The randomized peptides
are thus displayed on the external surface of the phage, where they can
bind to proteins or peptides. Phage binding to PDE3 proteins or peptides
may be separated from unbound phage using standard methods, for example,
by affinity chromatography to PDE3 peptides covalently linked to a solid
support such as a membrane or chromatography beads. If desired, it is
possible to collect bound phage, detach them from the PDE3 peptides by
exposure to an appropriate solution and proceed with another round of
binding and separation. This iterative process results in the selection
of phage with an increased specificity for PDE3.

[0258]Once phage of an appropriate binding stringency have been obtained,
it is possible to determine the amino acid sequence of the binding
peptide by sequencing the portion of the phage genome containing the
cDNA, for example, by using PCR primers that flank the cDNA insertion
site. Phage lacking any cDNA insert may be used as a control to ensure
that binding is specific.

[0259]The skilled artisan will realize that phage display may be used to
select for peptides (between 3 and 100, more preferably between 5 and 50,
even more preferably between 7 and 25, amino acid residues long) that can
bind to a desired protein or peptide. Such peptides may be of use, for
example, as potential inhibitors or activators of PDE3 catalytic activity
or protein-protein binding.

Methods for Screening Active Compounds

[0260]The present invention also contemplates the use of PDE3 isoform
proteins, peptides and active fragments, and nucleic acids encoding PDE3,
in the screening of potential PDE3 inhibitors or activators. These assays
may make use of a variety of different formats and may depend on the kind
of "activity" for which the screen is being conducted. Contemplated
functional "read-outs" include binding to a substrate (e.g., cAMP or
cGMP), inhibition of binding to a membrane or another protein,
phosphorylation or dephosphorylation of PDE3, or inhibition or
stimulation of a variety of cAMP-dependent processes, such as calcium
channel activation or protein kinase activity.

[0261]In Vitro Assays

[0262]In one embodiment, the invention is to be applied for the screening
of compounds that bind to the PDE3 isoforms or a fragment thereof. The
polypeptide or fragment may be either free in solution, fixed to a
support, or expressed in or on the surface of a cell. Either the
polypeptide or the compound may be labeled, thereby permitting the
determination of binding.

[0263]In another embodiment, the assay may measure the inhibition of
binding of PDE3 to a natural or artificial substrate or binding partner.
Competitive binding assays can be performed in which one of the agents is
labeled. Usually, the polypeptide will be the labeled species. One may
measure the amount of free label versus bound label to determine binding
or inhibition of binding.

[0264]Another technique for high throughput screening of compounds is
described in WO 84/03564, the contents of which are incorporated herein
by reference. Large numbers of small peptide test compounds are
synthesized on a solid substrate, such as plastic pins or some other
surface. The peptide test compounds are reacted with PDE3 and washed.
Bound polypeptide is detected by various methods.

[0265]Purified PDE3 can be coated directly onto plates for use in the
aforementioned drug screening techniques. However, non-neutralizing
antibodies to the polypeptide can be used to immobilize the polypeptide
to a solid phase. Also, fusion proteins containing a reactive region
(preferably a terminal region) may be used to link the PDE3 active region
to a solid phase.

[0266]Various cell lines containing wild-type or natural or engineered
mutations in PDE3 can be used to study various functional attributes of
these proteins and how a candidate compound affects these attributes.
Methods for engineering mutations are described elsewhere in this
document. In such assays, the compound would be formulated appropriately,
given its biochemical nature, and contacted with a target cell. Depending
on the assay, culture may be required. The cell may then be examined by
virtue of a number of different physiologic assays. Alternatively,
molecular analysis may be performed in which the function of PDE3 or
related pathways may be explored. This may involve assays such as those
for phosphorylation states of various molecules, cAMP levels, mRNA
expression for CREB-linked genes, or any other process regulated in whole
or in part by PDE3 activity. For certain embodiments, it may be desirable
to create "knock-out" cells that are lacking in endogenous
phosphodiesterase activity in order to specifically assay the effects of
various compounds on inserted isoforms of PDE3.

[0267]In Vivo Assays

[0268]The present invention also encompasses the use of various animal
models. By developing or isolating mutant cells lines that show
differential expression of one or more PDE3 isoforms, one can generate
animal models that will be predictive of cardiomyopathy and/or pulmonary
hypertension in humans and other mammals. These models may employ
transgenic animals that differentially express one or more PDE3 isoforms.

[0269]Treatment of animals with test compounds will involve the
administration of the compound, in an appropriate form, to the animal.
Administration will be by any route that could be utilized for clinical
or non-clinical purposes including, but not limited to, oral, nasal,
buccal, rectal, vaginal or topical. Alternatively, administration may be
by intratracheal instillation, bronchial instillation, intradermal,
subcutaneous, intramuscular, intraperitoneal, intravenous or
intra-arterial injection.

[0272]The goal of rational drug design is to produce structural analogs of
biologically active polypeptides or compounds with which they interact
(agonists, antagonists, inhibitors, binding partners, etc.). By creating
such analogs, it is possible to fashion drugs that are more active or
stable than the natural molecules, which have different susceptibility to
alteration or which may affect the function of various other molecules.
In one approach, one would generate a three-dimensional structure for
PDE3 or a fragment thereof. This could be accomplished by x-ray
crystallography, computer modeling based on the 3-D structures of other
phosphodiesterases or by a combination of both approaches. In addition,
knowledge of the polypeptide sequences permits computer-employed
predictions of structure-function relationships. An alternative approach,
an "alanine scan," involves the random replacement of residues throughout
a protein or peptide molecule with alanine, followed by determining the
resulting effect(s) on protein function.

[0273]It also is possible to isolate a PDE3-specific antibody, selected by
a functional assay, and then solve its crystal structure. In principle,
this approach yields a pharmacore upon which subsequent drug design can
be based. It is possible to bypass protein crystallography altogether by
generating anti-idiotypic antibodies to a functional, pharmacologically
active antibody. As a mirror image of a mirror image, the binding site of
an anti-idiotype antibody would be expected to be an analog of the
original antigen. The anti-idiotype could then be used to identify and
isolate peptides from banks of chemically or biologically produced
peptides. Selected peptides would then serve as the pharmacore.
Anti-idiotypes may be generated using the methods described herein for
producing antibodies, using an antibody as the antigen.

[0274]Thus, one may design drugs that have improved PDE3 isoform-selective
activity or that act as stimulators, inhibitors, agonists, or antagonists
of PDE3.

[0275]Knock-Out

[0276]The technique known as homologous recombination allows the precise
modification of existing genes, including the inactivation of specific
genes, as well as the replacement of one gene for another. Methods for
homologous recombination are described in U.S. Pat. No. 5,614,396,
incorporated herein by reference.

[0277]Homologous recombination relies on the tendency of nucleic acids to
base pair with complementary sequences. In this instance, the base
pairing serves to facilitate the interaction of two separate nucleic acid
molecules so that strand breakage and repair can take place. In other
words, the "homologous" aspect of the method relies on sequence homology
to bring two complementary sequences into close proximity, while the
"recombination" aspect provides for one complementary sequence to replace
the other by virtue of the breaking of certain bonds and the formation of
others.

[0278]First, a site for integration is selected within the host cell, such
as the PDE3A or PDE3B genes. Sequences homologous to the integration site
are included in a genetic construct, flanking the selected gene to be
integrated into the genome. "Flanking," in this context, simply means
that target homologous sequences are located both upstream (5') and
downstream (3') of the selected gene. The construct is then introduced
into the cell, permitting recombination between the cellular sequences
and the construct.

[0279]It is common to include within the construct a selectable marker
gene. This gene permits selection of cells that have integrated the
construct into their genomic DNA by conferring resistance to various
biostatic and biocidal drugs. In addition, this technique may be used to
"knock-out" (delete) or interrupt a particular gene. Thus, another
approach for inhibiting gene expression involves the use of homologous
recombination, or "knock-out technology." This is accomplished by
including a mutated or vastly deleted form of the heterologous gene
between the flanking regions within the construct. The arrangement of a
construct to effect homologous recombination might be as follows:
[0280]vector•5'-flanking sequence•selected
gene•selectable marker gene•flanking sequence-3'•vector

[0281]Using this kind of construct, it is possible, in a single
recombinatorial event, to (i) "knock out" an endogenous gene, (ii)
provide a selectable marker for identifying such an event, or (iii)
introduce a transgene for expression.

[0282]Another refinement of the homologous recombination approach involves
the use of a "negative" selectable marker. One example of the use of the
cytosine deaminase gene in a negative selection method is described in
U.S. Pat. No. 5,624,830. The negative selection marker, unlike the
selectable marker, causes death of cells that express the marker. Thus,
it is used to identify undesirable recombination events. When seeking to
select homologous recombinants using a selectable marker, it is difficult
in the initial screening step to identify proper homologous recombinants
from recombinants generated from random, non-sequence-specific events.
These recombinants also may contain the selectable marker gene and may
express the heterologous protein of interest, but will, in all
likelihood, not have the desired phenotype. By attaching a negative
selectable marker to the construct, but outside of the flanking regions,
one can select against many random recombination events that will
incorporate the negative selectable marker. Homologous recombination
should not introduce the negative selectable marker, as it is outside of
the flanking sequences.

Formulations and Routes for Administration to Patients

[0283]In certain embodiments, the isoform-selective inhibitors or
activators of PDE3 may be used for therapeutic treatment of medical
conditions, such as dilated cardiomyopathy and/or pulmonary hypertension.
Where clinical applications are contemplated, it will be necessary to
prepare pharmaceutical compositions in a form appropriate for the
intended application. Generally, this will entail preparing compositions
that are essentially free of pyrogens, as well as other impurities that
could be harmful to humans or animals.

[0284]Aqueous compositions of the present invention comprise an effective
amount of PDE3 inhibitor or activator, dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. Such compositions
also are referred to as innocula. The phrase "pharmaceutically or
pharmacologically acceptable" refers to molecular entities and
compositions that do not produce adverse, allergic, or other untoward
reactions when administered to an animal or a human. As used herein,
"pharmaceutically acceptable carrier" includes any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents and the like. The use of such media and
agents for pharmaceutically active substances is well known in the art.
Except insofar as any conventional media or agent is incompatible with
the PDE3 inhibitors or activators of the present invention, its use in
therapeutic compositions is contemplated. Supplementary active
ingredients also can be incorporated into the compositions.

[0285]The active compositions of the present invention may include classic
pharmaceutical preparations. Administration of these compositions
according to the present invention will be via any common route so long
as the target tissue is available via that route. This includes oral,
nasal, buccal, rectal, vaginal or topical. Alternatively, administration
may be by orthotopic, intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection. Such compositions normally
would be administered as pharmaceutically acceptable compositions.

[0286]The active compounds also may be administered parenterally or
intraperitoneally. Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water suitably
mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also
can be prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof and in oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.

[0287]The pharmaceutical forms suitable for injectable use include sterile
aqueous solutions or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be fluid to
the extent that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and fungi. The
carrier can be a solvent or dispersion medium containing, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), suitable mixtures thereof, and
vegetable oils. The proper fluidity can be maintained, for example, by
the use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants.

[0288]The prevention of the action of microorganisms can be brought about
by various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many
cases, it will be preferable to include isotonic agents, for example,
sugars or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.

[0289]Sterile injectable solutions are prepared by incorporating the
active compounds in the required amount in the appropriate solvent with
various other ingredients enumerated above, as required, followed by
filtered sterilization. Generally, dispersions are prepared by
incorporating the various sterilized active ingredients into a sterile
vehicle that contains the basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile powders
for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum-drying and freeze-drying techniques
that yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.

[0290]The compositions of the present invention may be formulated in a
neutral or salt form. Pharmaceutically acceptable salts include the acid
addition salts that are formed by reaction of basic groups with inorganic
acids such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with free acidic groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine, trimethylamine,
histidine, procaine and the like.

[0291]Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered in a
variety of dosage forms such as injectable solutions, drug release
capsules and the like. For parenteral administration in an aqueous
solution, for example, the solution should be suitably buffered if
necessary and the liquid diluent first rendered isotonic with sufficient
saline or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and intraperitoneal
administration.

[0292]In this connection, sterile aqueous media that can be employed will
be known to those of skill in the art in light of the present disclosure.
For example, one dosage could be dissolved in 1 ml of isotonic NaCl
solution and either added to 1000 ml of hypodermoclysis fluid or injected
at the proposed site of infusion (see, for example, Remington's
Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580).
Some variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose for the
individual subject. Moreover, for human administration, preparations
should meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics' standards.

Examples

[0293]The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of skill
in the art that the techniques disclosed in the Examples that follow
represent techniques discovered by the inventors to function well in the
practice of the invention and, thus, can be considered to constitute
preferred modes for its practice. However, those of skill in the art
should, in light of the present disclosure, appreciate that many changes
can be made in the specific embodiments that are disclosed and still
obtain a like or similar result without departing from the spirit and
scope of the invention.

[0296]The resulting PCR product contained a unique PDE3 DraIII site at the
5' end and a stop codon at the 3' end. The stop codon was flanked
upstream by a Flag epitope-coding sequence and downstream by an XhoI
site. The PCR products were subcloned into the pCRII vector (Invitrogen,
Carlsbad, Calif.) and isolated from this vector as DraIII/XhoI fragments.
XhoI/DraIII fragments containing the ORF sequence of PDE3A1 (SEQ ID
NO:14) upstream from the unique DraIII site were restricted from
pBluescript. In a three-way ligation, these 5' XhoI/DraIII fragments were
ligated via the DraIII site to the 3' DraIII/XhoI Flag epitope-containing
fragments and to XhoI-cut pZero vector (Invitrogen), to give PDE3A1
Flag-pZero. PDE3A1-Flag was then excised from pZero with XhoI, ligated
into pAcSG2 vector, subcloned and amplified.

[0299]Cytosolic and KCl-washed microsomal fractions, from the left
ventricular myocardium of explanted hearts of cardiac transplant
recipients with idiopathic dilated cardiomyopathy, were prepared by
homogenization, differential sedimentation and high-salt washing. Each
preparation was made from tissue pooled from at least three different
hearts. Tissue from left ventricular free walls was trimmed of epicardium
and endocardium, cut into roughly 0.5 cm3 pieces, rapidly frozen in
liquid nitrogen, and stored at -80° C. until use. To prepare
subcellular fractions, 0.3 g of the frozen tissue were added to five
volumes of buffer (5 mM KH2PO4/K2HPO4 and 2 mM EDTA (pH 6.8, 4°
C.), 1 mM dithiothreitol, 1 mM benzamidine, 0.8 mM PMSF, and 1 μg/ml
each of pepstatin A, leupeptin, and antipain). The tissue was homogenized
twice for ten seconds each. The homogenate was sedimented at 14,000 rpm
for 20 minutes using an Eppendorf Model 5415 centrifuge. The supernatant
was saved and the pellet resuspended in 1.5 ml of buffer, then
rehomogenized and resedimented in order to solubilize any trapped
cytosolic proteins. The supernatants containing cytosolic proteins were
pooled and diluted 1:1 with buffer containing 40% v/v glycerol and stored
at -80° C. until use. Comparable fractions of cultured human
aortic myocytes (Clonetics, East Rutherford, N.J.; seventh passage) were
similarly prepared.

and the complementary anti-sense primer (corresponding to nt 1435-76 of
the PDE3A1 ORF--SEQ ID NO:14) were used for mutagenesis. After
amplification in E. coli (XL1-Blue), mutated plasmids were purified using
a QIAprep Spin Miniprep kit (Qiagen, Valencia, Calif.) and sequenced.

[0304]PCR products with different five deletions were generated from the
wild-type and mutated pBluescript-PDE3A1 plasmids using five sense
primers containing T7 promoter sites immediately upstream from
gene-specific sequences and an anti-sense primer containing the stop
codon and a poly-A tail. The sense primers used for amplification in
these reactions were as follows:

[0306]In vitro translation products were synthesized from the PCR fragment
templates and labeled with 4 μCi [35S]methionine (1000 Ci/mmol)
in reticulocyte lysates using the TnT T7 Quick for PCR DNA system
(Promega, Madison, Wis.). To make a synthetic protein, a PCR product
containing a 5 deletion and a T7 promoter sequence was added to the TnT
T7 PCR Quick Master Mix and incubated for roughly 60 to 90 minutes at
30° C. This process was repeated for each PCR construct containing
a 5 deletion. Proteins thus created were isolated and subsequently
analyzed by autoradiography.

[0307]5' RACE

[0308]PCR amplification was performed on Marathon RACE-Ready cDNA from
human myocardium (Clontech, Palo Alto, Calif.) using 1 pmol gene-specific
anti-sense primer and 1 pmol sense primer corresponding to the 5' end of
the manufacturer's 5' tag. A second round of PCR was performed for 35
cycles using 1 pmol nested gene-specific primer and 1 pmol nested sense
primer corresponding to a second sequence within the manufacturer's tag.
RACE products were purified on agarose gels and ligated into the pCR2.1
vector with T4 ligase (14° C. overnight) using a TA cloning kit
(Invitrogen, San Diego, Calif.). Competent cells (INV F') were
transformed using a One Shot Kit (Invitrogen, Austin, Tex.) and plated on
X-gal LB-ampicillin plates (100 μg/ml ampicillin). Positive colonies
were grown overnight in LB-ampicillin medium. Plasmids were purified
using Mini- or Midiprep Plasmid purification systems (Qiagen) and inserts
were excised with EcoRI. Insert sizes were estimated by electrophoresis
through agarose gels.

[0309]Southern and Northern Blotting

[0310]DNA probes were prepared from PDE3A1 plasmid by PCR using
region-specific primers. PCR products were purified using QIA Quick Kits
(Qiagen). DNA was labeled with [32P]dCTP (3000 Ci/mmol, 10 mCi/ml)
using a random primer labeling kit (Stratagene). Unincorporated
nucleotides were removed using Sepahadex G-50 (fine) columns (Roche,
Indianapolis, Ind.). For Southern blotting, linear DNA corresponding to
nt (-)268 to nt 2610 of the PDE3A1 ORF (SEQ ID NO:14, SEQ ID NO:18) was
prepared from PDE3A1 template by PCR and purified as described above. The
PCR product was quantified by measurement of the A260/A280 ratio and its
purity confirmed by agarose gel electrophoresis. PCR product samples were
subjected to electrophoresis on 0.7% agarose gels, transferred to Gene
Screen Plus Nylon Membranes (New England Nuclear, Boston, Mass.),
cross-linked and pre-hybridized for two to three hours in QuikHyb
(Stratagene). Labeled DNA probes were hybridized with DNA blots at
65° C. for three to four hours using 1.25×106 cpm/ml of
probe and 0.1 mg/ml salmon sperm DNA. Following hybridization, excess
radiolabeled probe was removed by rinsing in SSC/0.1% SDS and
autoradiography was performed at -80° C. For Northern blotting,
RNA was extracted from human left ventricular myocardium from the excised
hearts of transplant recipients with dilated cardiomyopathy using TRI
reagent (Molecular Research Center, Cincinnati, Ohio). PolyA RNA was
prepared from total RNA using a Message Maker kit (Life Technologies,
Rockville, Md.). RNA was quantified and its purity confirmed as described
above. PolyA RNA samples were subjected to electrophoresis on 1% agarose
0.5 M formaldehyde gels, transferred to Gene Screen Plus Nylon Membranes,
cross-linked and pre-hybridized for two to three hours in QuikHyb.
Labeled DNA probes were hybridized with RNA blots, excess radiolabeled
probe was removed.

Example 2

PDE3 Isoforms in Cardiac and Vascular Myocytes

[0311]It has been shown that proteins of three different apparent
molecular weights can be immunoprecipitated from mammalian myocardium
with anti-PDE3 antibodies (Smith et al., 1993). These proteins are
identified herein as PDE3 isoforms by Western blotting of cytosolic and
microsomal fractions of human myocardium, using antibodies raised against
peptides derived from the PDE3A ORF.

[0312]An antibody against the C-terminus of PDE3 ("anti-CT") reacted with
three proteins in these fractions (FIG. 5). The largest, with an apparent
MW of 136,000 on SDS-PAGE ("PDE3A-136"), was present exclusively in
microsomal fractions (FIG. 5). Another PDE3 isoform, with an apparent MW
of 118,000 ("PDE3A-118"), was present in both microsomal and cytosolic
fractions, as was a third isoform with an apparent MW of 94,000
("PDE3A-94") (FIG. 5).

[0313]An antibody against an amino acid sequence between NHR2 and CCR
("anti-MID") reacted with PDE3A-136 and PDE3A-118 but not PDE3A-94 (FIG.
5). An antibody against amino acids 25-49 ("anti-NT") did not react with
any protein in microsomal or cytosolic fractions, indicating the absence
of this region from cardiac and vascular PDE3 isoforms (FIG. 5). However,
anti-NT did react with an rtPDE3A1 containing the full-length ORF (FIG.
5).

[0314]The antibodies were used to identify PDE3 isoforms in subcellular
fractions of aortic myocytes (Choi et al., 2001). Anti-CT reacted with
94-kDA and 118-kDa proteins in microsomal and cytosolic fractions of
aortic myocytes (not shown). Anti-MID reacted only with the 118-kDa
proteins (not shown). No proteins were visualized with anti-NT, and the
136-kDa protein band was absent in all cases (not shown).

[0315]Western blotting was used to show that PDE3B is present in vascular
myocytes, where it appears as a 137-kDa band in the microsomal fraction
(PDE3B-137) (Liu and Maurice, 1998). Western blots (not shown) indicate
PDE3B-137 is absent from myocardium (not shown). These results are
summarized in Table 3.

[0316]All three polyclonal antibodies (anti-NT, anti-MID and anti-CT)
reacted with recombinant PDE3A1. Anti-CT reacted with proteins in the
cytosolic and microsomal fractions of human myocardium that had apparent
molecular weights of 94,000 Da and 118,000 Da. Anti-CT also reacted with
a protein with an apparent molecular weight of 136,000 Da that was seen
only in microsomal myocardial fractions. Anti-MID also reacted with the
118,000 and 136,000 proteins, but not the 94,000 Da protein.

Example 3

Mechanisms for Generating Cardiac and Vascular PDE3A Isoforms

[0317]Addition of [35S]-labeled rtPDE3A (full-length ORF, SEQ ID
NO:14) to a sample of human myocardium prior to the preparation of
cytosolic and microsomal fractions provided no evidence for the
generation of smaller isoforms by proteolysis of the labeled full-length
rtPDE3A (not shown). Other potential mechanisms were investigated.

[0318]The migration of cardiac and vascular isoforms of PDE3A were
compared to those of recombinant proteins generated by in vitro
transcription/translation. PDE3A constructs were prepared with 5'
deletions designed to yield rtPDE3As starting from different in-frame
ATGs, inserted downstream from a T7 promoter and Kozak sequence (FIG. 3).
PDE3A-136, PDE3A-118 and PDE3A-94 migrated with the same apparent
molecular weights as the rtPDE3As starting at ATGs 1507, 1969 and 2521,
respectively (FIG. 6). This is consistent with the three PDE3A isoforms
being generated by transcription from alternative start sites.
Transcription/translation from every PDE3A-derived construct generated an
rtPDE3A whose apparent MW corresponded to PDE3A-94. To determine whether
the latter might be generated by translation from a downstream AUG, a
full-length rtPDE3A construct was prepared in which the ATG at nt 2521
was mutated to CTG (M to L). This mutation resulted in the disappearance
of rtPDE3A-94 (not shown). It is concluded that the PDE3A-94 isoform is
generated by transcription from the ATG initiation codon at nt 2521.

[0319]At least two different messenger RNA species are expressed in
different tissues: PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18) in cardiac
myocytes and PDE3A2 mRNA (SEQ ID NO:15) in both cardiac and vascular
myocytes (Choi et al., 2001). It appears that transcription from
alternative start sites in PDE3A results in the expression of PDE3A1 mRNA
(SEQ ID NO:14, SEQ ID NO:18) in cardiac myocytes and of PDE3A2 mRNA (SEQ
ID NO:15) in cardiac and vascular myocytes. From the above results, it is
concluded that PDE3A-136 is generated in cardiac myocytes by translation
from the second AUG in PDE3A1 mRNA (SEQ ID NO:14), while PDE3A-118 and
PDE3A-94 are generated in cardiac and vascular myocytes by translation
from alternative downstream AUGs in PDE3A2 mRNA (SEQ ID NO:15) (FIG. 7).

Example 4

Structure-Function Relationships in PDE3A Isoforms

[0320]FIG. 11 shows the complete amino acid sequence of the open reading
frame (ORF) for PDE3A. To date, three isoforms of PDE3A have been
characterized. These are apparently generated by N-terminal truncation of
the PDE3A ORF (SEQ ID NO:14). The apparent N-terminal methionine residues
of the three isoforms are indicated in bold in FIG. 11. Those are located
at residues 146 for PDE3A-136, 300 for PDE3A-118 and either 484 or 485
for PDE3A-94. The locations of the phosphorylation sites on the PDE3A
isoforms are indicated by underlining in FIG. 11. The P1 site is located
at residues 288-294, the P2 site at residues 309-312 and the P3 site at
residues 435-438. The P2 and P3 sites on the PDE3A isoforms contain a
single serine residue and the phosphorylated amino acid is unambiguous.
The P1 site contains multiple serine residues and it is presently unknown
which of these is covalently modified by phosphorylation.

Example 5

Functional Domains of PDE3A Isoforms

[0321]The functional domains in the cardiac and vascular isoforms of PDE3
are shown in Table 4. The domains were elucidated in part by comparison
of the electrophoretic migration, via SDS-PAGE, of native PDE3 isoforms
and recombinant PDE3A isoforms generated by in vitro
transcription/translation from constructs with 5 deletions of the open
reading frame designed to result in translation from different in-frame
start codons (ATG codon sequences). The rtPDE3A deletion constructs and
the locations of the different ATG start codons in the PDE3A1 ORF (SEQ ID
NO:14) are illustrated in FIG. 12. All recombinant isoforms migrated with
apparent molecular weights approximately 20,000 higher than predicted by
their amino acid sequences (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3).

[0322]The apparent molecular weight of PDE3A-136 was slightly higher than
the apparent molecular weight of 131,000 for the recombinant protein as
translated from ATG-2 in the PDE3A1 open reading frame (SEQ ID NO:14),
indicating that PDE3A1-136 contains part of the NHR1 site (a finding
consistent with its recovery only in microsomal fractions), all of NHR2,
and the P1, P2 and P3 sites for phosphorylation arid activation by PK-B
and PK-A. PDE3A-136 is, therefore, generated in cardiac myocytes from
PDE3A1, either by translation from ATG1 followed by targeted N-terminal
proteolysis or by some post-translational modification that reduces its
electrophoretic mobility, resulting in a higher apparent molecular
weight.

[0323]The apparent molecular weight of PDE3A-118 was indistinguishable
from that of the recombinant PDE3A translated from ATG4, indicating that
PDE3A-118 lacks NHR1 and the PK-B activation site, but includes the NHR2
and PK-A sites. PDE3A-118 is generated in cardiac and vascular myocytes
from PDE3A2 mRNA (SEQ ID NO:15) by translation from ATG4, the third ATG
in the open reading frame predicted by the cloned cDNA (GenBank Accession
No. NM000921), or by translation from ATG2 or ATG3 followed by targeted
N-terminal proteolysis.

[0324]The apparent molecular weight of PDE3A-94 was approximately equal to
the apparent molecular weight of 94,000 for the recombinant PDE3A
translated from ATG7/8, indicating that PDE3A-94 contains neither the
membrane-association domains NHR1 and NHR2, nor any of the three
phosphorylation sites. PDE3A-94 is generated in cardiac and vascular
myocytes from PDE3A2 mRNA (SEQ ID NO:15), either by translation from
AUG7/8 or by translation from a more upstream ATG followed by proteolytic
removal of a more extensive length of N-terminal sequence. That PDE3A-118
and PDE3A-94 are generated from a single mRNA (SEQ ID NO:15) by
alternative translational processing in vivo is consistent with the
observation that a PDE3A-94-like protein is generated from longer
constructs by translation from downstream ATG sequences in vitro.

[0325]The N-terminus predicted from the PDE3A1 open reading frame (SEQ ID
NO:14) was absent from native PDE3A-136, the longest PDE isoform
identified. All three isoforms contain the same C-terminal amino acid
sequences, downstream of different N-terminal starting points.

[0326]PDE3A-136 and PDE3B-137, which contain the transmembrane helices of
NHR1, would be expected to be exclusively membrane-bound in cardiac and
vascular myocytes. PDE3A-118, which contains NHR2 but not NHR1, and
PDE3A-94, which lacks both NHR1 and NHR2, would be expected to associate
reversibly with intracellular membrane proteins or to be partitioned
between the cytosolic and microsomal compartments. Their presence in both
microsomal and cytosolic fractions is compatible with this conclusion.
Further, the fact that PDE3A-118 and PDE3A-94 can be recovered in
microsomal fractions suggests that interactions with anchoring or
targeting proteins are involved in their intracellular localization.

[0327]The N-terminal sequence differences may cause different PDE3
isoforms to interact with different anchoring or targeting proteins that
localize them to different signaling modules. As a consequence, each PDE3
isoform may regulate the phosphorylation of different substrates of PK-A
and PK-G.

[0328]Surprisingly, transcription/translation from every PDE3A-derived
construct generated a recombinant PDE3A isoform whose apparent molecular
weight corresponded to that of PDE3A-94. Determination whether the latter
might be generated by translation from a downstream AUG in the
full-length PDE3A mRNA (SEQ ID NO:14, SEQ ID NO:18) was performed by
expression of a mutated construct starting from ATG1 in the PDE3A1 mRNA
(SEQ ID NO:14) in which ATG7/8 was mutated to CTGCTG (Met-Met Leu-Leu).
Expression of the mutated construct resulted in the disappearance of the
94,000 molecular weight recombinant PDE3A, a result consistent with the
generation of PDE3A-94 from the full-length PDE3 mRNA by translation from
AUG7/8.

Example 6

5 RACE PCR

[0329]Studies have shown that a PDE3A2 mRNA (SEQ ID NO:15), whose sequence
is identical to that of the PDE3A1 cDNA downstream of roughly nucleotide
300 in the latter's open reading frame (SEQ ID NO:14) but which lacks
PDE3A1's upstream sequence (SEQ ID NO:14, SEQ ID NO:18), is present in
both cardiac and vascular myocytes, while PDE3A1 mRNA (SEQ ID NO:14, SEQ
ID NO:18) is present in cardiac but not in vascular myocytes (Y. H. Choi
et al., Biochem J., 2001). To determine if the PDE3A2 mRNA (SEQ ID NO:15)
contained an alternative sequence upstream of roughly nucleotide 300, 5
RACE PCR was performed on a human myocardial cDNA library using three
pairs of anti-sense primers derived from the shared sequences of PDE3A1
(SEQ ID NO:14) and PDE3A2 (SEQ ID NO:15).

[0330]Subcloning and sequencing of the multiple 5 RACE products indicated
that the PDE3A2 mRNA (SEQ ID NO:15) contained no alternative sequence
upstream of roughly nucleotide 300 (not shown). Similar results were
obtained when 5 RACE was performed with comparable primers on a human
aortic cDNA library (not shown).

Example 7

Southern and Northern Blotting

[0331]Northern and Southern blotting was performed on nucleic acids from
human left ventricular myocardium using probes derived from different
regions of the PDE3A1 open reading frame (see SEQ ID NO:14). The first
nucleic acid probe, derived from nucleotides (-)268-189, corresponded to
a region predicted to be present in PDE3A1 (SEQ ID NO:14), but not in
PDE3A2 (SEQ ID NO:15). The other two nucleic acid probes used
corresponded, respectively, to nucleotides 517-957 and 2248-2610 of
PDE3A1 (SEQ ID NO:14), regions predicted to be present in both PDE3A1
(SEQ ID NO:14) and PDE3A2 (SEQ ID NO:15).

[0332]All three of the nucleic acid probes bound to an 8.2 kilobase band
(not shown). The two downstream probes also bound to a 6.9 kilobase band
to which the upstream probe did not bind (not shown). These results
indicate that the 8.2 kilobase band is PDE3A1 (SEQ ID NO:14, SEQ ID
NO:18) and the 6.9 kilobase band is PDE3A2 (SEQ ID NO:15). The size
differences observed between the two hybridized bands are accounted for
by the absence of the first roughly 300 nucleotides of the open reading
frame of PDE3A1 (SEQ ID NO:14) from PDE3A2 (SEQ ID NO:15), consistent
with the generation of the latter by alternative transcription or
splicing within exon 1 of the open reading frame of PDE3A1 (SEQ ID
NO:14). This result is consistent with a result predicted by ribonuclease
protection assays of RNA prepared from human myocardium and cultured
human aortic myocytes with antisense probes spanning nucleotides 208-537
and nucleotides 2248-2610 of PDE3A1 (SEQ ID NO:14) (Y. H. Choi et al.,
Biochem J., 2001). Importantly, PDE3A1 mRNA (SEQ ID NO:14, SEQ ID NO:18)
and PDE3A-136 were determined to be present in only cardiac myocytes
while PDE3A2 mRNA (SEQ ID NO:15), PDE3A-118, and PDE3A-94 were present in
both cardiac and vascular myocytes. This result indicates that PDE3A1
mRNA (SEQ ID NO:14, SEQ ID NO:18) gives rise to PDE3A-136 and PDE3A2 mRNA
(SEQ ID NO:15) gives rise to both PDE3A-118 and PDE3A-94.

Example 8

Inhibitors of PDE3 Activity and Effects of Intracellular Localization on
Catalytic Activity

[0333]The effects of two PDE3 inhibitors, cilostazol (not shown) and
milrinone (FIG. 8), on cAMP hydrolytic activity in cytosolic and
microsomal fractions of human myocardium were examined. These drugs had
more potent effects in microsomal fractions (FIG. 8).

[0334]The PDE3 inhibitor milrinone was used to quantify PDE3 cAMP- and
cGMP-hydrolytic activity in lysates of Sf9 cells expressing recombinant
PDE3A isoforms and in cytosolic and salt-washed microsomal fractions of
human myocardium (Table 5). Catalytic activity was measured at 0.1 μM
cAMP and cGMP. Milrinone-sensitive activity for tissue fractions was
calculated by measuring cyclic nucleotide hydrolysis inhibited by
milrinone at concentrations equal to its IC50 values for cAMP and
cGMP hydrolysis by recombinant PDE3A1, and dividing by 0.5.

[0335]The ratio of milrinone-sensitive cAMP/cGMP hydrolytic activity in
cytosolic fractions was lower than that observed with full-length
recombinant PDE3A, while the ratio in microsomal fractions was higher. To
determine if these differences were the result of N-terminal deletions,
the same studies were performed on lysates of Sf9 cells expressing a
recombinant PDE3A from which the N-terminus had been deleted
(rtPDE3AΔ5). N-terminal truncation did not affect the cAMP/cGMP
activity ratio.

[0336]The higher ratio of milrinone-sensitive cAMP/cGMP hydrolytic
activity in the microsomal fraction relative to that seen in recombinant
PDE3 isoforms suggests that localization to intracellular membranes
increases the selectivity of PDE3 isoforms for cAMP, possibly resulting
from the interaction of membrane-bound PDE3 with other proteins.

[0337]The contribution of PDE3 isoforms to compartmental regulation of
cyclic nucleotide hydrolysis was examined in subcellular preparations
from native human myocardium and cultured pulmonary artery myocytes. The
results are presented in Table 6.

[0338]PDE3 comprises the majority of the total cAMP hydrolytic activity in
microsomal fractions of human myocardium at both low and high cAMP
concentrations. It comprises a smaller but significant fraction of cAMP
hydrolytic activity in cytosolic fractions of these cells, probably
reflecting the larger presence of other cAMP phosphodiesterases in the
cytosol. In cultured pulmonary artery myocytes, these findings are
reversed. PDE3 contributes less to membrane-bound cAMP hydrolytic
activity but more to cytosolic cAMP hydrolytic activity.

[0339]PDE3 comprises a large portion of the total cGMP hydrolytic activity
in microsomal fractions of human myocardium at low but not at high cGMP
concentrations. This likely reflects the presence of other lower affinity
cGMP phosphodiesterases in these fractions.

[0340]PDE3 contributes relatively little to cGMP hydrolytic activity in
cytosolic fractions of human myocardium. PDE3 comprises a surprisingly
small portion of the total cGMP hydrolytic activity in both microsomal
and cytosolic fractions of pulmonary artery myocytes at both low and high
concentrations of substrate. The fact that PDE3s contribute less to total
cGMP hydrolytic activity than to total cAMP hydrolytic activity in
subcellular fractions of these cells, taken in the context of the fact
that competitive PDE3 inhibitors inhibit cAMP hydrolytic activity more
potently than they inhibit cGMP hydrolytic activity of PDE3 (see Table
7), suggest that the clinical effects of currently available competitive
PDE3 inhibitors are likely to be mediated to a greater degree by
increases in cAMP content than by increases in cGMP content in both
cardiac and vascular myocytes. This conclusion cannot be extrapolated to
agents that may inhibit PDE3 activity through non-competitive mechanisms
proposed herein. The latter may change the profile of cellular actions of
PDE3 inhibition, representing an additional possible benefit to the
approaches proposed over currently available therapies.

Phosphorylation Sites and Effects of Phosphorylation on PDE3A Isoforms

[0341]The phosphorylation sites on PDE3A-136 were localized by labeling
studies to amino acid residues 288-294 (P1 site), 309-312 (P2 site) and
435-438 (P3 site). The P2 and P3 sites on PDE3A-136 only contain one
serine residue each and the phosphorylated residue is unambiguous (FIG.
11). The P1 site contains multiple serine residues and it is not certain
at present which is phosphorylated.

[0342]Differences with respect to the presence of PK-A and PK-B sites in
the different isoforms of PDE3 indicate differences in regulation by
phosphorylation. PDE3A-136 and PDE3B-137 contain sites P1, P2 and P3 and
are thus potentially subject to regulation by both PK-A and PK-B (Table
4). PDE3A-118 contains only P2 and P3 and can thus be regulated only by
PK-A (Table 4). PDE3A-94 contains none of these phosphorylation sites,
therefore, its activity can be regulated by neither PK-A nor PK-B. These
N-terminal sequence differences may lead to differences in regulation by
other interacting partners.

[0343]The effects of phosphorylation at the P3 site of PDE3A, along with
the apparently equivalent site on PDE3B-137, on phosphodiesterase
catalytic activity are shown in Table 8. Flag-tagged rtPDE3B-137 isoforms
(full ORFs) were prepared with mutations at P3, one of the two PK-A sites
(FIG. 4). These included a constitutively nonphosphorylated form, in
which Ser421 was mutated to alanine ("S421A") and a form that acted as if
it were constitutively phosphorylated, in which Ser421 was mutated to
aspartic acid ("S421D"). The charged group on the end of the aspartate
side chain resembles a phosphate group in its effect on phosphodiesterase
activity. These recombinant isoforms were used, together with the
corresponding wild-type rtPDE3B, to examine the effects of
phosphorylation at site P3 on catalytic activity and inhibitor
sensitivity. Catalytic activity of PDE3 was measured in
detergent-solubilized lysates of Sf21 cells expressing Flag-tagged rtPDE3
isoforms (full-length ORFs). Values for Vmax and Km were
calculated by nonlinear regression (first-order kinetics). Preparations
were diluted so that each contained equal concentrations of
immunoreactive PDE3 as determined by quantitative Western blotting with
anti-Flag antibodies. The three isoforms of rtPDE3B were observed to have
comparable catalytic activity toward cAMP and cGMP (Table 8). The three
isoforms also exhibited similar sensitivity to inhibition by cilostazol
(data not shown). This suggests that phosphorylation at P3 has little, if
any, direct effect on enzyme activity.

[0344]The rtPDE3Bs were used to study the effects of phosphorylation by
PK-A at other sites. Phosphorylation of these isoforms with PK-A caused a
much greater stimulation of activity in S421D than in S421A or the
wild-type rtPDE3B (FIG. 9). These results indicate an interaction between
P3 and P2, the upstream PK-A site. Phosphorylation of P3 may increase the
stimulation of activity by PK-A by facilitating phosphorylation at P2.
The fact that stimulation of the wild-type rtPDE3B has less effect than a
Ser→Asp mutation at P3 may reflect incomplete phosphorylation of
the latter site. Alternatively, phosphorylation of P3 may potentiate the
effect of phosphorylation of P2 on enzyme activity. Another possibility
is that phosphorylation of P3 has an inhibitory effect on catalytic
activity that is overcome by phosphorylation of P2.

Example 10

Site-Specific Mutations and Phosphorylation

[0345]The phosphorylation of PDE3B-137, PDE3A-136 and PDE3A-118 by PK-A
and PK-B is examined using recombinant constructs with thrombin cleavage
sites followed by his6 tags at the C-terminus. Constructs are
expressed in Sf9 cells by infection with baculovirus vector.
His6-tagged recombinant proteins are purified by Co2+-affinity
chromatography (Clontech resin) and their his6 tags are removed by
thrombin cleavage.

[0346]rtPDE3s are phosphorylated by PK-A (Sigma) and PK-B (Upstate
Biotechnology). Varying concentrations of purified rtPDE3s are incubated
in the presence of nanomolar concentrations of kinase, saturating
concentrations of [γ-32P]ATP and phosphatase inhibitors.
Reaction mixtures are subjected to SDS-PAGE, and 32P incorporation
is quantified in excised PDE3 bands following established protocols
(Movsesian et al., 1984). Values for Km and Vmax for
phosphorylation by PK-A and PK-B are calculated by nonlinear regression
and standardized using peptide substrates as controls (Kemptide for PK-A,
Crosstide for PK-B).

[0347]The use of rtPDE3s with Ser→Ala and Ser→Asp mutations
at selected phosphorylation sites allows the isolation of individual
sites (by rendering others nonphosphorylatable). Interactions between
sites may also be examined. For example, to study the effect of
phosphorylation at P2 by PK-A on phosphorylation at P1 by PK-B, rtPDE3s
are prepared with Ser∝3Ala and Ser→Asp mutations at P2 and
P3. The effects on Km and Vmax for phosphorylation by PK-B at
P1 are examined.

[0348]Non-physiologic artifacts may be induced using Ser→Asp
mutations. For example, they may mimic phosphorylation at a site that is
not phosphorylated in vivo in the cell of interest. To address this
problem, the phosphorylation of specific sites in. aortic myocytes and
HL-1 cells transfected with tagged rtPDE3s is examined. To examine
phosphorylation at P1, HL-1 cells are transfected with PDE3 constructs
with Ser→Ala and Ser→Asp mutations at P2 and P3, using HL-1
cells transfected with Ser→Ala mutations at P1 as a negative
control. Cells are preincubated with 32PO43- and exposed
to β1- and β2-adrenergic receptor agonists,
forskolin, PGE2 and IBMX (to activate PK-A) and/or IGF-1±wortmannin
(to activate PI3-K, which phosphorylates and activates PK-B). PDE3 is
immunoprecipitated from the resulting cellular fractions with anti-Tag
antibodies and subjected to SDS-PAGE and autoradiography to determine
whether phosphorylation at P1 has occurred and is influenced by
phosphorylation at other sites. Quantitative Western blotting is then
performed to normalize 32P incorporation to immunoreactive PDE3.
This approach may be used to determine whether phosphorylation of one
site affects phosphorylation of another in vivo (cultured cells). This
approach has been validated in adipocytes where the sites phosphorylated
in transfected proteins have been determined to be the same as those
phosphorylated in native proteins (Kitamura, et al., 1999).

[0349]Two similar approaches may be performed to validate phosphorylation
in cultured myocytes. First, antibodies are raised to synthetic peptides
corresponding to phosphorylated P1, P2 and P3 domains. The studies
described above are repeated in non-transfected cells (without
radiolabeling). SDS-PAGE is performed on cell homogenates and the
phosphor-specific antibodies are used to confirm or refute
phosphorylation at individual sites by Western blotting. The same studies
may be performed after preincubation with 32PO43-. Native
PDE3s are immunoprecipitated from cellular homogenates with anti-CT
antibodies. SDS-PAGE is performed on these native proteins and the PDE
bands are excised. The protein is extracted from the gel material and
limited proteolysis with trypsin, chymotrypsin, CNBr and/or V8 is
performed. The resulting peptide fragments are resolved via
two-dimensional mapping, using two-dimensional peptide maps of
mutagenized rtPDE3s phosphorylated in vitro as controls. Comparison
thereof reveals which sites are phosphorylated in the HL-1 cells.

Example 11

Effects of Phosphorylation on Intracellular Localization

[0350]The role of the N-terminus in intracellular targeting was elucidated
through an approach that involved the transfection of cultured cells with
rtPDE3 constructs. This approach may be expanded by stably transfecting
cultured aortic myocytes (Clonetics) with his6- or Flag-tagged
PDE3B-137- and PDE3A-118-derived constructs with Ser→Ala and
Ser→Asp mutations at the three phosphorylation sites identified
herein. PDE3A-94 is not included because it does not appear to contain
any of the phosphorylation sites.

[0351]The protocol for stable transfection uses the vector pCDNA 3.1
(Invitrogen). This vector is driven by a CMV promoter, includes a
neomycin resistance element for selection and adds a C-terminal
myc-his6 tag to the expressed protein. The choice of stable rather
than transient transfection is based on the higher levels of recombinant
protein expression observed in stable transformants (not shown). The
intracellular localization of rtPDE3 isoforms is determined by indirect
immunofluorescence using fluorophore-tagged anti-his6 or anti-Flag
antibodies. Co-localization relies on the use of antibodies to markers
for different intracellular membranes. Phosphorylation does not induce
translocation of PDE3B-137, as it contains the transmembrane helices of
NHR1 and is, therefore, likely to be an intrinsic membrane protein.
However, some combinations of Ser→Asp mutations induce a
translocation of PDE3A-118 from intracellular membranes to the cytosol.

[0352]The results of these studies may not be applicable to cardiac
myocytes, since the PDE3 isoforms are not identical and the intracellular
targeting mechanisms may differ. For this reason, the studies described
above may be repeated in cardiac myocytes or cells derived from cardiac
myocytes using PDE3A-136 instead of PDE3B-137.

Example 12

Indirect Immunofluorescence and Intracellular Localization

[0353]The effects of phosphorylation of the sites P1, P2 and P3 on the
membrane targeting domains NHR1 and NTIR2 and intracellular localization
were studied. The role of the N-terminus of PDE3 in intracellular
targeting was elucidated by transfecting cultured cells with rtPDE3
constructs and visualizing the intracellular localization of these rtPDE3
constructs by indirect immunofluorescence. COS-7 cells were transfected
with PDE3A and PDE3B constructs with C-terminal Flag-tags and varying
N-terminal deletions, and localization was visualized using
fluorescein-labeled anti-Flag antibodies. Constructs containing NHR1 were
found to be membrane-bound (not shown). Constructs lacking NHR1 but
containing NHR2 were partially membrane-bound and partially cytosolic and
constructs lacking both NHR1 and NHR2 were exclusively cytosolic (not
shown). This distribution corresponds to the distribution of native PDE3s
in human myocardium and aortic myocytes.

[0354]To extend this approach, cultured aortic myocytes (Clonetics, East
Rutherford, NJ) may be transfected with Flag-tagged PDE3B-137- and
PDE3A-118-derived constructs with Ser→Ala and Ser→Asp
mutations at the P1, P2 and P3 PK-A and PK-B phosphorylation sites.
Stable transfection utilizes the transcription vector pCDNA 3.1
(Invitrogen, Carlsbad, Calif.). The pCDNA vector is driven by a CMV
promoter, includes a neomycin resistance element for selection, and adds
a C-terminal Flag tag to the expressed protein. The intracellular
localization of rtPDE3 isoforms with mutagenized phosphorylation sites
may be determined by indirect immunofluorescence using fluorophore-tagged
anti-Flag antibodies. Co-localization relies on the use of commercially
available antibodies to markers for different intracellular membranes.

[0355]Results in vascular myocytes may not be applicable to cardiac
myocytes. The PDE3 isoforms in the two cell types are not identical, and
the intracellular targeting mechanisms may be different. For this reason,
the above studies may be repeated in HL-1 cells, an immortalized cell
line derived from atrial myocytes (Claycomb, et al., 1998). Western
blotting indicates that the representation of PDE3 isoforms in
subcellular fractions prepared from these cells is similar to that seen
in preparations from human left ventricular myocardium, making these
cells particularly suitable for these experiments. Transfections of HL-1
cells is performed with PDE3A-136- rather than PDE3B-derived constructs
to reflect the different patterns of cellular expression. This
transfection may be transient or stable. A high percentage of
transfection efficiency with PDE3 constructs using transient transfection
obviates the need for stable transfection of rtPDE3 isoforms.

Example 13

Protein-Protein Interactions

[0356]The interactions of PK-B with PDE3B were examined. Microsomal
fractions of 3T3 adipocytes (which express PDE3B) were solubilized with
NP-40 and fractionated by gel filtration. Western blotting showed the
presence of separate peaks for PDE3B and PK-B, but some of the PK-B was
found in the PDE3B peak (not shown). An association between PK-B and
PDE3B was confirmed by the ability of anti-PDE3B antibodies to
co-immunoprecipitate the two proteins in the PDE3B peak (not shown).
Treatment with insulin increased the phosphorylation of PK-B and appeared
to increase the percentage of PK-B co-purifying with PDE3B (not shown).
These results suggest that PK-B and PDE3B form stable complexes in vivo,
either by direct interaction or by co-interaction with another protein.

[0357]Detergent-solubilized lysates of Sf9 cells expressing rtPK-B were
mixed with detergent-solubilized lysates of Sf9 cells expressing one of
two Flag-tagged forms of PDE3B. The first isoform of PDE3B contained its
full ORF. The second lacked the N-terminal 604 amino acids containing the
NHR1, NHR2 and the three phosphorylation sites. PK-B could be
co-immunoprecipitated with anti-Flag antibodies in the presence of the
full-length rtPDE3B but not in the presence of the N-terminal-deleted
form (FIG. 10), confirming the role of the N-terminus of PDE3B in its
association with PK-B.

[0358]The addition of Flag-tagged rtPDE3B to 3T3 lysates allowed the
co-immunoprecipitation of AKAP220, which co-localizes PK-A and PP1
(Schillace et al., 2001). This indicates that interactions with other
proteins serves to localize PDE3 to specific signaling modules, and
suggests that blocking these interactions will alter the function of
PDE3.

[0359]Purified rtPDE3s may be used as affinity ligands to identify
PDE3-binding proteins ("PDE3-BPs") by interaction cloning from
phage-displayed myocardial and vascular smooth muscle cDNA libraries.
This approach involves two basic steps: preparation of phage-displayed
cDNA libraries and biopanning with rtPDE3.

[0360]Preparation of Phage-Displayed cDNA Libraries

[0361]cDNA inserts from commercially available human cardiac
(XbaI-(dT)15-primed) and aortic (oligo(dT) and random-primed)
libraries (Clontech, Palo Alto, Calif.) are PCR-amplified using
vector-derived primers (λTriplEx for cardiac, λgt10 for
aortic) with unique restriction sites. These libraries have been used to
clone PDE3 isoforms, which are expressed in relatively low abundance. PCR
products are size-fractionated on agarose gels. Products greater than 500
nucleotides in length are purified by agarose gel electrophoresis and
ligated into the genes of phage coat proteins using unique restriction
sites. Proteins or protein fragments encoded by the cDNA inserts are
displayed on the phage surface.

[0362]Two phages with different reproductive biologies are used. One is
M13, a non-lytic phage that is secreted after assembly in the bacterial
periplasm. cDNA inserts up to 1000 amino acids in length can be expressed
as C-terminal fusions to the pVI coat protein of M13. The protocols used
are as disclosed in Fransen et al. (1999). The same vectors and protocols
are used to insert human cardiac and aortic cDNA libraries into pVI. The
second phage is T7 (Novagen, Madison, Wis.). This phage, being lytic, is
processed quite differently from M13, so that cDNA inserts that may
interfere with M13 function are not likely to affect T7 (and vice versa).
T7 is capable of displaying cDNA products up to 1200 amino acids in
length. Methods for its use have been disclosed in Zozulya et al. (1999).

[0363]Biopanning with rtPDE3

[0364]Phages with cDNA inserts are incubated with rtPDE3s that are
immobilized either directly onto polystyrene wells or indirectly by
binding of C-terminal his6 tags to anti-his6 mAb, followed by
immunoprecipitation. Phage whose cDNA inserts encode full-length or
truncated PDE3-BPs are co-immobilized with PDE3, then eluted and
amplified in E. coli. Each round of this procedure yields a phage library
enriched in cDNAs encoding PDE3-binding proteins. Biopanning is repeated
through several iterations until the titer of phage binding to
immobilized PDE3 is ten-fold above background (phage binding to wells in
the absence of PDE3), at which point, individual phage colonies are
cloned and their cDNA inserts sequenced.

[0365]Phages are biopanned with rtPDE3s. rtPDE3A-118 and rtPDE3A-94 are
used for both cardiac and aortic libraries. PDE3B-137 and PDE3A-136 are
used exclusively for aortic and cardiac libraries, respectively.
Thiophosphorylated rtPDE3 are prepared with PK-A and/or PK-B and
ATPγS for use as bait in parallel experiments to select proteins
that bind preferentially to phosphorylated PDE3s, for example,
phosphatases. Phosphothioesters are resistant to dephosphorylation and
thiophosphorylated proteins, therefore, bind stably to protein
phosphatases.

[0366]Cloned cDNA sequences identified by biopanning may be used to search
protein databases and identify full-length binding proteins for PDE3.

[0367]The skilled artisan will realize that the methods discussed above
could be used to identify novel isoform-selective inhibitors or
activators of PDE3. Purified isoform proteins are used as ligands for
biopanning general phage display libraries comprising random nucleic acid
sequences encoding short peptides. Phages that bind with relatively high
affinity to one or more PDE3 isoforms are selected and their DNA inserts
are sequenced. The encoded peptides are chemically synthesized and their
ability to activate or inhibit PDE3 catalytic activity or to block or
mimic the effect of phosphorylation at P1, P2 or P3 on catalytic activity
is examined using standard enzyme analysis. The effect of identified
activators or inhibitors on each PDE3 isoform is determined and
isoform-selective compounds are identified. Use of site-specific
mutagenized isoforms that are designed to be constitutively
unphosphorylatable or to mimic constitutively phosphorylated residues at
P1, P2 and P3 identifies activators or inhibitors that are selective for
phosphorylated or dephosphorylated variants of each isoform.

Example 15

Characterization of Binding Interactions and Effects on PDE3 Function

[0368]Confirmation of Binding of Cloned Prospective PDE3-BPs to PDE3

[0369]Binding interactions are confirmed by co-immunoprecipitation, which
can occur in any of four ways. First, native PDE is immunoprecipitated
from lysates of cardiac and aortic myocytes using anti-PDE antibodies and
co-immunoprecipitation is confirmed via Western blotting using antibodies
raised to the cloned PDE-BP. The second method reverses the order of the
antibodies used. Thus, antibodies to the cloned PDE3-BP are used for
immunoprecipitation and co-immunoprecipitation is confirmed via Western
blotting using anti-PDE3 antibodies. Third, aortic myocytes or HL-1 cells
are transfected with Flag-tagged rtPDE3-BPs, followed by
co-immunoprecipitation and Western blotting with anti-Flag antibodies.
Lastly, tagged rtPDE3s and rtPDE3-BPs are expressed by in vitro
transcription/translation in reticulocyte lysates or in a baculovirus/Sf9
system. The recombinant proteins are co-incubated and
co-immunoprecipitation is tested for AKAP-220, a method described
elsewhere in this document.

[0370]Characterization of Binding Interactions and Effects on PDE3
Function

[0371]The affinity (KD) of the interaction between PDE3 and various
binding proteins or peptides may be determined by ELISA, using
immobilized rtPDE3 and rtPDE3-BPs (obtained by expression in E. coli or
Sf9/St21 cells). The effects of rtPDE3-BPs on the catalytic activity and
inhibitor sensitivity of rtPDE3s is determined as described above. The
effects of PDE3-BPs on the phosphorylation of rtPDE3s by PK-A and PK-B in
vitro is determined as described above. rtPDE3s with Ser→Ala and
Ser→Asp mutations are used to determine how phosphorylation at
specific sites affects interactions with PDE3-BPs.

[0372]Interacting domains of PDE3s and their binding partners are
identified by deletional and site-directed mutagenesis of PDE3 and/or
PDE3-BPs. Peptides derived from interacting domains are examined for
inhibition of PDE3/PDE3-BP interactions. Inhibition of PDE3/PDE3-BP
interactions is examined by ELISA or by measuring inhibition of
functional correlates of binding. For example, if binding to a PDE3-BP
increases the Km of PDE3 for cAMP, the ability of peptides to
prevent this increase is determined. Alternatively, peptides that mimic
the effects of PDE3-BPs may be PDE3 activators. Peptides in either
category are of interest as potential therapeutic agents and may serve as
templates for peptidomimetic drugs or reporters for high-throughput
screening.

[0373]Peptides derived from the phase display experiments derived above
are also tested for their ability to either block the binding of PDE3 to
PDE3-BPs or to mimic the effect of PDE3-BPs on catalytic activity or
inhibitor sensitivity of PDE3.

[0374]To quantify the affinity of PDE3 to PDE3-BP, surface plasmon
resonance (Biacore, Piscataway, NJ) using purified rtPDE3s and rtPDE3-BPs
(obtained by expression in E. coli or Sf9/Sf21 cells) is performed.
Generally, surface plasmon resonance (SPR) uses light reflected from a
conducting film at the interface between two media of different
refractive index. In this instance, the media are the biological sample
and the glass of a sensor chip. The conducting film is a thin layer of
gold on the sensor chip surface. When the molecules in the biological
sample bind to the surface of the sensor chip, the concentration (and,
therefore, the refractive index) at the chip surface changes and an SPR
response is detected. Here, his-tagged rtPDE3s are captured by anti-his
monoclonal antibodies immobilized on flow-cell surfaces of biosensor
chips. A series of concentrations of rtPDE3-BPs (expressed in Sf9 cells
and purified as described above) are superfused thereon and surface
plasmon resonance responses are used to determine values for KD.

[0375]Effects of Phosphorylation on Interactions Between PDE3 and PDE3-BP

[0376]To determine the effects of phosphorylation at specific sites on
interactions between PDE3 and PDE3-BP, surface plasmon resonance
experiments are performed as above using rtPDE3s with Ser→Ala and
Ser→Asp mutations at the three phosphorylation sites P1, P2 and
P3. The effects of these mutations on the KD of the reaction
described above are determined. The kinetics of phosphorylation at P1, P2
and P3 by PK-A and PK-B in the presence and absence of PDE3-BPs are also
determined.

[0377]The ability of any new PDE3 kinase to phosphorylate P1, P2 and P3
may be examined for PK-A and PK-B, as described above.

[0378]The ability of PDE3 phosphatases to dephosphorylate P1, P2 and P3
may also be determined. This entails the use of rtPDE3's with
Ser→Ala mutations at all but one of the phosphorylation sites.
These rtPDE3s are phosphorylated in the presence of [γ-32P]ATP
and the appropriate kinase (e.g., PK-A or PK-B). 32P release in the
presence of phosphatase is characterized in terms of Vmax and
Km. rtPDE3s with Ser→Asp mutations are then used to determine
the effect of phosphorylation at one site or dephosphorylation at
another.

[0379]The effect of PDE3-BP's interactions on catalytic activity,
substrate preference, and inhibitor sensitivity is determined by
measuring cyclic nucleotide hydrolysis in the absence and presence of
PDE3-BPs. Functional K values for PDE3/PDE3-BP's interactions are
determined and compared to the KD values determined by surface
plasmon resonance.

[0380]Identification of the Interacting Domains of PDE3 and PDE3-BP

[0381]Identification of the interacting domains of PDE3s and PDE3-BPs is
done via deletional and site-directed mutagenesis of PDE3 and/or PDE3-BP.
Several lines of evidence suggest that compartmentally nonselective
increases in intracellular cAMP content in cardiac myocytes have both
beneficial and harmful effects in dilated cardiomyopathy. Agents capable
of selectively activating or inhibiting individual PDE3 isoforms
localized to different intracellular compartments or of selectively
affecting activity toward cAMP or cGMP may offer major advantages in
therapeutic applications. Peptides that block or interfere with the
interaction of PDE3 with PDE3-BP may be used to identify functional
consequences in vivo. Alternatively, peptides that mimic the effects of
PDE3-BPs may be PDE3 activators. Either category of peptides would be
useful tools for studying the function of PDE3 isoforms in vivo and may
be of interest as prototypical therapeutic agents. They may serve as
templates for peptidomimetic drugs or may be tagged for use as reporters
for high throughput screening.

Example 16

siRNA Inhibition of PDE3A1

[0382]21-nucleotide siRNAs are chemically synthesized using Expedite RNA
phosphoramidites and thymidine phosphoramidite chemistries (Proligo,
Germany). Synthetic oligonucleotides are deprotected and gel-purifed. The
siRNA sequence targeting the PDE3A1 mRNA corresponds to the nucleotide
sequences -268 to -241 of the human myocardial PDE3A1 cDNA sequence (SEQ
ID NO:18; GenBank Accession No. NM000921). That sequence is located in
the 5' untranslated region of the PDE3A1 mRNA (SEQ ID NO:18) and is not
present in PDE3A2 (SEQ ID NO:15). It should, therefore, be specific for
inhibition of expression of the PDE3A-136 protein.

[0383]Sf21 cells expressing rtPDE3A1 are grown at 37° C. in TNM-FH
media (BD-Pharmingen, San Diego, Calif.). Transfection with 1.0 nM siRNA
is performed with Oligofectamine (Life Technologies) as described by the
manufacturer. Cells are incubated 20 hours after transfection and
expression of rtPDE3A1 is assayed by Northern blotting. Transfection with
siRNA is observed to result in a complete inhibition of rtPDE3A1
expression in Sf21 cells. Control cells are transfected with a random 21
by siRNA sequence and show no affect on rtPDE3A1 expression.

Example 17

Isoform-Specific Probe and Antisense Construct

[0384]In certain embodiments of the invention, isoform-specific probes may
be constructed and used, for example, to determine the levels of
expression of the PDE3 isoforms in different cells or tissues or in
response to various putative inhibitors or activators, such as in a
high-throughput screening assay directed towards mRNAs. Because the
downstream (3') portions of the PDE3A mRNAs (SEQ ID NO:14, SEQ ID NO:15)
are apparently identical, the only region available for isoform-specific
probes and/or antisense constructs are at the 5' end of the PDE3A1 mRNA
(SEQ ID NO:14, SEQ ID NO:18). An exemplary probe specific for the mRNA
encoding the PDE3A-136 isoform protein is disclosed below:

[0385]The probe sequence corresponds to nucleotides -268 to 189 of PDE3A1
(SEQ ID NO:14, SEQ ID NO:18), where nucleotide 1 starts with the first
ATG codon in the largest open reading frame (ORF) of the PDE3A1 cDNA
sequence (SEQ ID NO:14). The probe sequence (SEQ ID NO:13) is located
primarily in exon 1 of the PDE3A1 mRNA, starting in the 5' UTR and ending
just before the NHR1 sequence. Primers may be used to generate the probe
from the PDE3A1 cDNA or to amplify the target sequence from sample RNA,
as disclosed below:

[0386]The skilled artisan will realize that there are many potential uses
for the isoform-specific probe and primers disclosed above. For example,
expression of PDE3A1 could be measured in various cells or tissues in
either normal individuals or individuals with a disease state, such as
cardiomyopathy and/or pulmonary hypertension. The effects of various
putative activators or inhibitors on PDE3A1 expression in intact cells
could be determined as part of a high-throughput screening assay.
Alternatively, an antisense construct, ribozyme and/or siRNA inhibitor
could be designed to bind only to PDE3A1 mRNA (SEQ ID NO:14, SEQ ID
NO:18). Such an inhibitor would decrease activity of PDE3A-136, while
leaving PDE3A-118 and PDE3A-94 activity unaffected. Since SEQ ID NO:13
shows the sequence of part of the PDE3A1 cDNA, the skilled artisan will
realize that an antisense construct would be designed to be
complementary, preferably exactly complementary, to part or all of the
sequence of SEQ ID NO:13. Such a construct could be designed as a
double-stranded DNA sequence that is functionally coupled to a promoter
and inserted into an expression vector that can be transfected into a
target cell. Expression vectors of use in mammalian cells are well known
in the art, as summarized above.

[0387]All of the compositions, methods and apparatus disclosed and claimed
herein can be made and executed without undue experimentation in light of
the present disclosure. While the compositions and methods of this
invention have been described in terms of preferred embodiments, it will
be apparent to those of skill in the art that variations may be applied
to the compositions, methods and apparatus and in the steps or in the
sequence of steps of the methods described herein without departing from
the concept, spirit and scope of the invention. More specifically, it
will be apparent that certain agents that are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All such
similar substitutes and modifications apparent to those skilled in the
art are deemed to be within the spirit, scope and concept of the
invention as defined by the appended claims.